Systems, compositions, and methods for transplantation

ABSTRACT

Systems and methods for purification and concentration of autologous alpha-2-macroglobulin (A2M) from whole blood are provided. Also provided are diagnostic methods for identifying sites in the synovial joints, spine, tendons or ligaments for treatment of pain, degeneration, or inflammation with autologous A2M. Methods for utilizing autologous A2M in combination with other autologous treatments (e.g. platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half life of the protein in joints and spine disc or epidural space.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/380,234, filed on Aug. 21, 2014, pursuant to 35 U.S.C. § 371 as aUnited States National Phase Application of International ApplicationNo. PCT/US2013/027159, filed Feb. 21, 2013, which claims the benefit ofU.S. Provisional Application No. 61/601,434, filed on Feb. 21, 2012,U.S. Provisional Application No. 61/726,815, filed on Nov. 15, 2012,U.S. Provisional Application No. 61/726,840, filed on Nov. 15, 2012,U.S. Provisional Application No. 61/727,433, filed on Nov. 16, 2012, andU.S. Provisional Application No. 61/740,218, filed on Dec. 20, 2012,which applications are incorporated herein by reference in theirentirety.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND OF THE INVENTION

Inflammation causing spinal and joint pain can be difficult to treat.Increasing degrees of inflammation and force applied to joints result injoint injury. Abnormal joint anatomy can be a hallmark of aging, butjoint injury can be also a result of trauma, such as chondral lesionsoften seen in athletes. While joint injury resulting from trauma can betypically associated with acute inflammation, aberrant joint anatomyresulting from aging (e.g., osteoarthritis) can be a chronic condition.Physicians currently do not have a system or method available todifferentiate between acute injury due to trauma and age related jointdeteriorations.

Presently, it can be difficult to determine the appropriate course oftreatment for a given patient since it can be frequently unclear whetherthe particular condition the patient suffers from may be acute orchronic or if pathology in the joint is the cause of the pain.

Spinal-related pain can be typically classified as discogenic,facetogenic or radiculopathic pain. The manifestation of radiculopathicpain has traditionally been attributed to various physical and/ormechanical abnormalities, such as compression or mechanical irritationof the nerve root related to conditions such as disc herniation,stenosis, spondylolisthesis, sciatica, piriformis syndrome, obturatorsyndrome, cystic lesions (e.g., ganglion and synovial), tumors, andother pathology, such as chemically mediated causes.

Numerous studies have attempted to elucidate the pathophysiology ofspinal-related pain, and several molecular pathways have been implicatedtentatively. However, no clear causal pathway leading from injury ordegeneration to the painful state has been confirmed. Molecular markerscan be linked to clinical symptoms, and serve as potential targets forthe development of diagnostics and therapeutic tools. Although somestudies have provided evidence that the epidural space can be affectedby an intervertebral disc herniation, none has measured concentrationsof biomolecules in the epidural space in an attempt to detect thedifferences between affected and non-affected persons.

Tendons, which connect muscle to bone, and ligaments, which connectbones to other bones, are both composed of bands of fibrous connectivetissue. The cells of the fibrous connective tissue are mostly made up offibroblasts the irregular, branching cells that secrete strong fibrousproteins (such as collagens, reticular and elastic fibers, andglycoproteins) as an extracellular matrix. The extracellular matrix canbe defined in part as any material part of a tissue that is not part ofany cell. So defined, the extracellular matrix (ECM) can be thesignificant feature of the fibrous connective tissue.

The ECM's main component can be various glycoproteins. In most animals,the most abundant glycoprotein in the ECM can be collagen. Collagen canbe tough and flexible and gives strength to the connective tissue.Indeed, the main element of the fibrous connective tissue is collagen(or collagenous) fiber. The ECM also contains many other components:proteins such as fibrin and elastin, minerals such as hydroxyapatite, orfluids such as blood plasma or serum with secreted free flowingantigens. Given this diversity, it can serve any number of functions,such as providing support and anchorage for cells (which attach viafocal adhesions), providing a way of separating the tissues, andregulating intercellular communication. Therefore, the ECM can functionin a cell's dynamic behavior.

Injury to tendons and ligaments causes damage not only to the connectivetissue, but to the extracellular matrix as well. Damage to the ECM caninterrupt cell behavior in the connective tissue and decrease and/orlimit healing. After injury, continuing damage can be caused byproduction of matrix metalloproteinases (MMPs) by the body. MMPs areenzymes that degrade all components of the ECM. This can lead to animbalance between the synthesis and degradation of the ECM, as the bodytries to heal itself while the enzymes remodel the ECM. An overabundanceof remodeling by MMPs cause damage to previously connected tissue whichresults in the formation of scar tissue. In addition, scar tissueadhesion to surrounding tissue can cause further pulling and/orstretching of the tendons or ligaments and resultant pain.

Currently, treatment of injury to tendons and ligaments includes somesimple measures such as: avoiding activities that aggravate the problem;resting the injured area; icing the area the day of the injury; andtaking over-the-counter anti-inflammatory medicines. However, thesesimple remedies do not always cure the injury and often more advancedtreatments are needed. These treatments include: corticosteroidinjections, platelet-rich plasma (PRP), hyaluronic acid (HA) injection,physical therapy and even surgery. Corticosteroids are often usedbecause they can work quickly to decrease the inflammation and pain.Physical therapy can include range of motion exercises and splinting(such as for the fingers, hands, and forearm). Surgery can be onlyrarely needed for severe problems not responding to the othertreatments. It can be appreciated that additional treatment measures areneeded to treat and prevent extracellular matrix degradation for quickerand improved healing of tendons and ligaments.

Alpha-2-macroglobulin (A2M) is a highly conserved protease inhibitorpresent in plasma at relatively high concentrations (0.1-6 mg/ml). It isunique in its ability to inhibit all the major classes of proteases(Bhattacharjee et al (2000) J. Biol. Chem. 275, 26806-26811). A2M can beproduced by several cell types, such as hepatocytes, lung fibroblasts,macrophages, astrocytes and tumor cells (Borth W, “Alpha2-macroglobulin, A multifunctional binding and targeting protein withpossible roles in immunity and autoimmunity,” Ann. N.Y. Acad. Sci.737:267-272 (1994)). A2M often exists as a tetramer of four identical180 kDa subunits that forms a hollow cylinder-like structure. It canpresent multiple target peptide bonds to attacking proteases in itscentral “bait” domain. A2M can be the major protease inhibitor acting onforeign proteases, such as snake venoms. However, there are many otherprotease inhibitors in the circulation and it has been proposed that A2Mcan have other functions including binding to and regulation of cytokineand growth factor activity, promotion of tumoricidal capabilities ofmacrophages, and enhancement of antigen presentation. A2M can also be atargeting carrier for cytokines or growth factors.

Therefore, it is an object of the invention to provide compositions,systems, methods, and kits for the detection, diagnosis, and treatmentof inflammation, pain in the spine or joint, degradation ofextracellular matrix, and inhibiting fibronectin aggrecan complex (FAC)(FIG. 1). It is another object of the invention to provide biomarkersand methods for identifying sites in the spine or joint for treatingpain. It is another object of the invention to provide biomarkers thatcan be used to diagnose or assist in the diagnosis be of the presence ofpathologies that are causative of spinal- or joint-related pain. It isanother object of the invention to provide methods for diagnosing orassisting in the diagnosis of the presence of pathologies that arecausative of spinal- or joint related pain. Yet another object of theinvention is to provide biomarkers and methods to determine anappropriate therapy for a subject experiencing spinal- or joint-relatedpain. Another object of the invention is to provide biomarkers andmethods to monitor and assess the efficacy of a treatment for spinal- orjoint-related pain. Another object of the invention is to providecompositions and methods for treating spinal or joint pain and forselecting treatment sites in the spine or joint for treatment to inhibitor reduce pain.

It is another object of the invention to provide variant polypeptidesfor treating inflammation and pain. It is another object of theinvention to provide variant A2M polypeptides that inhibit the formationof fibronectin aggrecan complex (FAC). It is another object of theinvention to provide variant A2M polypeptides with a higher proteaseinhibitory activity than a wild-type A2M polypeptide. It is anotherobject of the invention to provide methods of making variantpolypeptides for the treatment of inflammation and pain.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a liquid composition comprising: (a)alpha-2-macroglobulin (A2M) isolated from a biological sample from amammal, wherein the A2M is present at a concentration of at least 1.1times higher than the concentration of A2M present in the biologicalsample from the mammal; and (b) plasma, bone marrow aspirate (BMA), oranother body fluid from the biological sample. In some embodiments, anycomposition provided herein further comprises proteins with a molecularweight higher than 500 kDa wherein the proteins with a molecular weighthigher than 500 kDa are present at a concentration of at least 1.1 timeshigher than found in the biological sample from the mammal. In someembodiments, the concentration of molecules with a molecular weight lessthan 500 kDa is less than 90%, 70%, 50%, 30%, or 10% of theconcentration of those proteins and/or fold concentration of A2M presentin the biological sample from the mammal. In some embodiments, themolecules with a molecular weight less than 500 kDa comprise cytokines;chemokines; other immunomodulatory mediators including peptides,proteins, DNA, RNA, carbohydrates and small molecules; proteases; andother degradative proteins with a molecular weight of less than 500 kDa.In some embodiments, the cytokines comprise interleukins, tumor necrosisfactors (TNFs), monocyte chemoattractant proteins (MCPs), macrophageinflammatory proteins (MIPs), tumor growth factors (TGFs), and matrixmetalloproteases (MMPs). In some embodiments, the concentration of A2Mpresent in the biological sample is between about 0.1 mg/mL to 6 mg/mL.In some embodiments, the biological sample is a blood sample, BMA, orother body fluid. In some embodiments, any of the compositions providedherein further comprise one or more additional non-blood derivedcomponents. In some embodiments, the one or more additional non-bloodderived components comprise an anti-coagulant, wherein theanti-coagulant comprises EDTA, tri-sodium citrate, water for injection(WFI), or saline. In some embodiments, any of the compositions providedherein further comprise one or more additional blood-derived components.In some embodiments, the one or more additional blood-derived componentscomprise platelets. In some embodiments, the composition issubstantially free of cells and particles larger than 1 μm, andcomprises a reduced concentration of proteins and other molecules with amolecular weight of 500 kDa or less compared to the biological sample.In some embodiments, the composition is for autologous delivery into oneor more joints of the mammal, and wherein the one or more joints areselected from the synovial, diarthrodial, amphiarthrodial,synarthrodial, symphyseal, and cartilaginous joint. In some embodiments,the mammal is a human. In some embodiments, the A2M is present at aconcentration of at least 1.5 times higher than the concentration of A2Mpresent in the biological sample from the mammal. In some embodiments,the A2M is present at a concentration of at least 2 times higher thanthe concentration of A2M present in the biological sample from themammal, optionally wherein the A2M is present at a concentration of atleast 3, 5, 10, or 20 times higher than the concentration of A2M presentin the biological sample from the mammal. In some embodiments, any ofthe compositions provided herein further comprise platelets.

In one aspect, provided herein is a method for enrichment of A2M from asample obtained from a mammal comprising: (a) flowing the sample throughone or more filters, thereby separating the sample into a filtrate and aretentate; and (b) collecting the retentate, wherein the retentate isenriched for A2M and wherein the concentration in said retentate ofproteins having a molecular weight of less than about 500 kDa is lessthan 90% of the concentration of those proteins and/or foldconcentration of A2M in the sample. In some embodiments, theconcentration of A2M in the retentate is at least 1.1, 1.5, 2, 3, 4, 5,10, or 20 times higher than the concentration of A2M in the mammaliansample. In some embodiments, the retentate comprises less than 90%, 80%,60%, 30%, or 10% fold concentration of A2M and/or concentration of theproteins with a molecular weight less than about 500 kDa from themammalian sample. In some embodiments, the mammalian sample comprisesplasma. In some embodiments, red blood cells and white blood cells havebeen removed from the mammalian sample. In some embodiments, themammalian sample further comprises one or more blood derived components.In some embodiments, the one or more blood derived components compriseplatelets. In some embodiments, red blood cells, white blood cells, andplatelets are removed from the mammalian sample. In some embodiments,the red blood cells, white blood cells, and platelets are removed byflowing or passing the mammalian sample through the one or more filters.In some embodiments, the one or more filters is characterized by havinga pore size of at most 0.1 μm, 0.6 μm, 1 μm, or higher. In someembodiments, the one or more filters is a hollow fiber tangential flowfilter, is characterized by having a molecular weight cut-off of at most500 kDa, or a combination thereof. In some embodiments, the one or moreother filters comprise a charge, immobilized molecules, or a combinationthereof, thereby enhancing the selectivity of the one or more filters.In some embodiments, the immobilized molecules comprise antibodies,proteins, receptors, ligands, carbohydrates, nucleotides, RNA, or DNA.In some embodiments, enhancing the selectivity of the one or morefilters comprises enhancing the ability of the one or more filters toretain A2M, enhancing the ability of the one or more filters to notretain molecules that are not A2M, or a combination thereof. In someembodiments, flowing the sample through one or more filters comprisesapplying tangential force filtration, one or more centrifugation steps,gravitational forces, mechanical forces, or any combination thereof. Insome embodiments, the mechanical force comprises a pump, centrifugalforce, gas pressure, or a force that can flow a liquid through the oneor more filters. In some embodiments, any of the methods provided hereinfurther comprise adding one or more non-blood derived components, one ormore blood derived components, or a combination thereof, to themammalian sample before or during step (a), to the retentate after step(a), or both. In some embodiments, the one or more additional non-bloodderived components comprises an anti-coagulant, preservative, excipient,diluent, or other additive. In some embodiments, the anti-coagulantcomprises EDTA, tri-sodium citrate, water for injection (WFI), saline,or ACD-A. In some embodiments, the diluent is a WFI solution or a salinesolution. In some embodiments, the one or more additional blood derivedcomponents comprise platelets. In some embodiments, the retentate issubstantially free of cells and particles larger than 0.1 μm, 0.2 μm,0.6 μm, and/or 1 μm and comprises a reduced concentration of proteinsand other molecules with a molecular weight of 500 kDa or less comparedto the A2M concentration in the biological sample. In some embodiments,the mammalian sample is from a human subject. In some embodiments, thehuman subject has a disease or condition treatable with the retentate.In some embodiments, the diseases or conditions treatable with theretentate comprise cancer, degenerative diseases, traumatic diseases,and/or inflammatory diseases, whose pathogenesis includes the activityof proteases. In some embodiments, the cancer, degenerative diseases,traumatic diseases, and/or inflammatory diseases whose pathogenesisincludes the activity of proteases comprises osteoarthritis,inflammatory arthritides, enthesopathies, tendinopathies, ligamentousinjuries, and degenerative diseases of the bone, cartilage, tendons, andligaments, post operation of tendons, wound healing, and othermusculoskeletal diseases. In some embodiments, the biological sample iscollected with the aid of an additional absorbent, adsorbent, orcapillary materials or devices selected from the group of needle-syringecombo, sponges, wicks, pledgets, sutures, hydrophilic catheters,hydrophobic catheters, hollow-lumen catheters, or any combinationthereof.

In one aspect, provided herein is a method for enrichment of A2M from amammalian sample comprising: (a) flowing or passing the sample throughone or more first filters, thereby separating the sample into a firstfiltrate and a first retentate; (b) flowing the first filtrate throughone or more second filters, thereby separating the sample into a secondfiltrate and a second retentate enriched in A2M; and (c) collecting thesecond retentate. In some embodiments, the one or more first filters arecharacterized by having a pore size of at most 0.1 μm, 0.6 μm, or 1 μm.In some embodiments, the one or more second filters are characterized byhaving a molecular weight cut-off of at most 500 kDa. In someembodiments, the retentate is obtained in less than about 15 minutes, 30minutes, 45 minutes, 1 hour, or 3 hours.

In one aspect, provided herein is a system for enrichment of A2M from amammalian sample comprising: (a) one or more filters; and (b) acentrifuge, a pump, or a combination thereof, wherein cells, particles,and other molecules larger than 1 μm and proteins with a molecularweight of less than about 500 kDa are removed from the sample by flowingthe sample through the one or more filters in sequence. In someembodiments, the flow filtration module is a dead end and/or tangentialflow filtration module. In some embodiments, any system provided hereinfurther comprises one or more waste modules. In some embodiments, thesample is flowed or passed through the one or more filters in sequenceby applying centrifugal force, using the pump, or a combination thereof;thereby producing an A2M enriched retentate. In some embodiments, anysystem provided herein further comprises a collection module, whereinthe A2M enriched retentate is collected after passing the sample throughthe one or more filters. In some embodiments, cells, particles, andother molecules larger than 0.6 μm and proteins with a molecular weightof less than about 500 kDa removed from the sample by flowing the samplethrough the one or more filters in sequence are deposited into the oneor more waste modules. In some embodiments, any system provided hereinfurther comprises a sample loading module operable to introduce thesample into the system. In some embodiments, the sample loading moduleis directly or indirectly attached to the blood stream of a subject.

In one aspect, provided herein is a system for concentrating A2M from afluid sample comprising: a flow filtration module comprising an inlet,an outlet, and two or more filters; wherein the two or more filters arefluidly connected in series between the filter unit inlet and outlet;wherein a flow of fluid sample passes through the at least two filtersto produce an A2M concentrated serum; wherein a first of the two or morefilters screens out cells, particles, and other molecules larger than0.1 μm; and wherein a second of the two or more filters retain moleculesof weight more than about 500 kDa. In some embodiments, the flowfiltration module is a dead end and/or tangential flow filtrationmodule. In some embodiments, any system provided herein furthercomprises a pump adapted to be fluidly coupled to the filtration moduleeither upstream of the inlet or downstream of the outlet of thefiltration module, said pump further adapted to produce a flow of thefluid sample that passes through the filter unit from the inlet to theoutlet. In some embodiments, the first and the second of two or morefilters comprise a first and a second cross flow filter. In someembodiments, the filter module further comprises a first and a secondpermeate collection reservoir, and wherein the first permeate collectionreservoir stores a permeate from the first cross flow filter and aretentate of the first cross flow filter, and wherein the secondpermeate collection reservoir stores a permeate from the second crossflow filter and the concentrated A2M from serum or plasma comprises aretentate of the second cross flow filter. In some embodiments, theconcentrated A2M from serum or plasma remains in the first permeatecollection reservoir. In some embodiments, the retentate of the firstcross flow filter remains in a collection bag. In some embodiments, thefirst permeate from the first cross flow filter flows through the secondcross flow filter. In some embodiments, any system provided hereinfurther comprises a centrifuge and/or centrifugation step. In someembodiments, the first and the second of two or more filters comprise afirst and a second cross flow filter.

In one aspect, provided herein is a system for concentrating A2M from afluid sample comprising: a filtration module comprising an inlet, anoutlet, and one or more filters; wherein the one or more filters arefluidly connected in series between the filter module inlet and outlet;wherein a flow of the fluid sample passes through the one or morefilters to produce a concentrated A2M serum; wherein a first of the oneor more filters screens out cells, particles, and other molecules largerthan 1 μm; and wherein a second of the one or more filters retainsmolecules of weight more than about 500 kDa. In some embodiments, anysystem provided herein further comprises a pump adapted to be fluidlycoupled to the filtration module either upstream of the inlet ordownstream of the outlet of the filtration module, said pump furtheradapted to produce a flow of the fluid sample that passes through one ormore filters of the filter module. In some embodiments, the first andthe second of two or more filters comprise a first and a second crossflow filter. In some embodiments, the filter module further comprises afirst and a second permeate collection reservoirs, and wherein the firstpermeate collection reservoir stores a permeate from the first crossflow filter, and wherein the first permeate flows through the secondcross flow filter and a retentate of the first cross flow filter willremain in a first retentate collection reservoir, and wherein the secondpermeate collection reservoir stores a permeate from the second crossflow filter and the retentate of the second cross flow filter comprisesconcentrated A2M the fluid sample.

In one aspect, provided herein is a system for concentrating A2M from afluid sample comprising: a centrifuge; a filtration module comprising aninlet, an outlet, and one or more filters; and a supernatant of thefluid sample obtained from by centrifuging the fluid sample with thecentrifuge, wherein the one or more filters are fluidly connected inseries between the filter module inlet and outlet, wherein a flow of thefluid sample passes through the one or more filters to produce aconcentrated A2M serum, wherein the flow of the fluid sample that passesthrough the filtration module comprises the supernatant of the fluidsample, wherein the one or more filters of the filtration modulecomprise at least one 500 kDa cross flow filter configured to retainmolecules of weight more than about 500 kDa in a retentate reservoir,wherein the permeate from the 500 kDa cross flow filter is collected ina permeate reservoir, and wherein the retentate of the 500 kDa crossflow filter comprises concentrated A2M.

In one aspect, provided herein is a method of concentrating A2M in afluid sample comprising: providing a filtration module, wherein thefiltration module comprises an inlet, an outlet, and one or more filtersfluidly connected in series between the inlet and outlet; pumping thefluid sample through the filtration module inlet, the one or morefilters and the outlet to produce a concentrated A2M serum, whereinpumping the fluid sample is accomplished with a pump fluidly connectedto the filtration module either upstream of the inlet or downstream ofthe outlet; and removing cells from the fluid sample, wherein at leastone 500 kDa filter of the one or more filters retains molecules ofweight more than about 500 kDa. In some embodiments, removing cells fromthe fluid sample comprises providing a centrifuge, centrifuging thefluid sample, and obtaining a resultant supernatant of the fluid sample.In some embodiments, removing cells from the fluid sample comprisespumping the fluid sample through a first filter of the filtrationmodule, wherein the filter screens out cells, particles and othermolecules larger than 1 μm. In some embodiments, the first filtercomprises a first cross-flow filter and the at least one 500 kDa filtercomprises a second cross-flow filter. In some embodiments, any of themethods provided herein further comprise filtering a permeate of thefirst cross-flow filter with the second cross-flow filter; and retaininga retentate of the second cross-flow filter, wherein the retentate ofthe second cross flow filter comprises concentrated A2M. In someembodiments, any of the methods provided herein further comprise storingthe retentate of the second cross-flow filter containing theconcentrated A2M in a second cross-flow filter retentate reservoir. Insome embodiments, any of the methods provided herein further comprisestoring the retentate of the first-cross flow filter in a firstcross-flow filter retentate reservoir. In some embodiments, any of themethods provided herein further comprise retaining a pellet of thecentrifuged fluid sample.

In one aspect provided herein is a method of concentrating A2M in afluid sample comprising: providing a flow filtration module, wherein thefiltration module comprises an inlet, an outlet, and two or more filtersfluidly connected in series between the inlet and outlet; and pumpingthe fluid sample through the filtration module inlet, the two or morefilters and the outlet to produce a concentrated A2M serum or plasma,and wherein pumping the fluid sample is accomplished with a pump fluidlyconnected to the filtration module either upstream of the inlet ordownstream of the outlet, and wherein a first of the two or more filtersscreens out cells, particles, and other molecules larger than 0.1 μm,and wherein a second of the two or more filters retain molecules ofweight more than about 500 kDa. In some embodiments, the flow filtrationmodule is a dead end and/or tangential flow filtration module. In someembodiments, the first and second of the at least two filters comprise afirst and a second cross flow filter. In some embodiments, thefiltration module further comprises: retaining a permeate of the firstcross flow filter in the first permeate collection reservoir; andpassing a permeate of the first cross flow filter to the second crossflow filter; and retaining a permeate of the second cross flow filter ina second permeate collection reservoir, wherein the concentrated A2Mfrom serum or plasma comprises a retentate of the second cross flowfilter in a collection bag. In some embodiments, any of the methodsprovided herein further comprise centrifuging the fluid sample to removecells and particles, thereby forming plasma or serum, and placing theplasma and/or serum into the collection bag.

In one aspect, provided herein is a method of treating a subject,comprising administering to a subject in need thereof an effectiveamount of any composition described herein or a composition obtainableby any method described herein. In some embodiments, the composition isadministered into an anatomic site relevant to a pathology of thesubject. In some embodiments, protease activity is inhibited at ananatomic site of administration; thereby decreasing the degenerationrate of tissue, the degeneration rate of cartilage, the degenerationrate of discs, or synovial inflammation, or a combination thereof. Insome embodiments, the subject has one or more conditions comprising:arthritis, inflammation, ligament injury, tendon injury, bone injury,cartilage degeneration, cartilage injury, an autoimmune disease, backpain, joint pain, joint degeneration, disc degeneration, spinedegeneration, bone degeneration, or any combination thereof; whereininflammation comprises joint or disc inflammation caused by surgery,joint or disc inflammation caused by a joint or disc replacement, or acombination thereof. In some embodiments, the subject has beenpreviously diagnosed with the one or more conditions. In someembodiments, the administration is to a joint selected from the groupcomprising a wrist, spinal, shoulder, elbow, carpal, metacarpal,phalangeal, acromioclavicular, sternoclavicular, scapular, costal,sacroiliac, hip, knee, ankle, tarsal, and a metatarsal joint.

In one aspect, provided herein is a method of inhibiting the formationor causing the dissociation of the fibronectin-aggrecan complex (FAC) ina subject with a condition comprising administering an agent to thesubject, wherein the agent inhibits one or more proteins or cellsassociated with formation of the FAC, thereby inhibiting FAC formation.In some embodiments, the condition comprises cancer, arthritis,inflammation, ligament injury, tendon injury, bone injury, cartilagedegeneration, cartilage injury an autoimmune disease, back pain, jointpain, joint degeneration, disc degeneration, spine degeneration, bonedegeneration, inflammation in joint or disc surgery, inflammation injoint or disc replacement, or any combination thereof. In someembodiments, the agent comprises an antibody, polypeptide, nucleotide,or small molecule. In some embodiments, the agent binds to the FAC butnot to the individual components of the complex separately. In someembodiments, the agent comprises a recombinant aggrecan G3 domain,wherein the domain contains the aggrecan G3 Lectin domain andcompetitively binds to fibronectin; and wherein the newly formed complexlacks the binding site to Pathogen Associated Molecular Patterns (PAMP)receptor and the binding site Damage Associated Molecular Patterns(DAMP) receptor. In some embodiments, the agent comprises a recombinantfibronectin fragment, wherein the fragment comprises a G3 binding domainand competitively binds to aggrecan, and wherein the newly formedfibronectin fragment aggrecan G3 complex lacks the binding site to PAMPreceptor, and the DAMP receptor. In some embodiments, the agentcomprises an aggrecan antibody. In some embodiments, the agent comprisesa fibronectin antibody. In some embodiments, the agent comprises anantibody that binds to the PAMP receptor recognition domain of aggrecan,the DAMP receptor recognition domain of aggrecan, or both, therebyinhibiting activation of monocytes and other cells. In some embodiments,the agent comprises an antibody that binds to the PAMP receptorrecognition domain of fibronectin, the DAMP receptor recognition domainof fibronectin, or both, thereby inhibiting activation of monocytes andother cells. In some embodiments, the agent comprises a PAMP receptor orDAMP receptor that binds to the PAMP domain of aggrecan G3, the DAMPdomain of aggrecan G3, or both, thereby inhibiting activation ofmonocytes and other cells. In some embodiments, the agent comprises asoluble form of the PAMP receptor or DAMP receptor that binds to thePAMP domain of fibronectin, the DAMP domain of fibronectin, or both,thereby inhibiting activation of monocytes and other cells. In someembodiments, the agent inhibits production of proinflammatory cytokines,chemokines, proteases, or any combination thereof. In some embodiments,the agent inhibits fibroblast cells, thereby inhibiting production offibronectin, recruitment of other fibroblast cells, or a combinationthereof. In some embodiments, the small molecule or polypeptide isidentified using one or more high-throughput screening methods. In someembodiments, the small molecule or polypeptide inhibits FAC formation,causes the dissociation of FAC, inhibits activation of monocytes,inhibits increased production of fibronectin, inhibits recruitment offibroblast cells, binds to the DAMP domain of fibronectin, binds to theDAMP domain of aggrecan G3, binds to the PAMP domain of fibronectin, orbinds to the PAMP domain of aggrecan G3. In some embodiments, the smallmolecule or polypeptide inhibits FAC formation by competitively bindingto fibronectin or aggrecan. In some embodiments, the small molecule orpolypeptide binds to the FAC complex resulting in dissociation ordegradation of the FAC complex. In some embodiments, inhibiting theformation of the fibronectin-aggrecan complex (FAC) comprises inhibitingof one or more steps in FAC formation. In some embodiments, the one ormore steps in FAC formation comprise production of fibronectin in theECM, production of proteases and metalloproteases, production ofinflammatory cytokines and chemokines, degradation of aggrecan incartilage, and production of aggrecan G3 domain fragment.

In one aspect, provided herein is an agent for use in therapy, whereinsaid agent inhibits the formation of the fibronectin-aggrecan complex(FAC) in a subject with a condition, and wherein the agent inhibits oneor more proteins or cells associated with formation of the FAC, therebyinhibiting FAC formation. In some embodiments, the agent comprises anantibody, polypeptide, nucleotide, or small molecule. In someembodiments, the agent binds to the FAC but not to the individualcomponents of the complex separately. In some embodiments, the agentcomprises a recombinant aggrecan G3 domain, wherein the domain containsthe aggrecan G3 Lectin domain and competitively binds to fibronectin;and wherein the newly formed complex lacks the binding site to PathogenAssociated Molecular Patterns (PAMP) receptor and the binding siteDamage Associated Molecular Patterns (DAMP) receptor. In someembodiments, the agent comprises a recombinant fibronectin fragment,wherein the fragment comprises a G3 binding domain and competitivelybinds to aggrecan, and wherein the newly formed fibronectin fragmentaggrecan G3 complex lacks the binding site to PAMP receptor, and theDAMP receptor. In some embodiments, the agent comprises an aggrecanantibody. In some embodiments, the agent comprises a fibronectinantibody. In some embodiments, the agent comprises an antibody thatbinds to the PAMP receptor recognition domain of aggrecan, the DAMPreceptor recognition domain of aggrecan, or both, thereby inhibitingactivation of monocytes and other cells. In some embodiments, the agentcomprises an antibody that binds to the PAMP receptor recognition domainof fibronectin, the DAMP receptor recognition domain of fibronectin, orboth, thereby inhibiting activation of monocytes and other cells. Insome embodiments, the agent comprises a PAMP receptor or DAMP receptorthat binds to the PAMP domain of aggrecan G3, the DAMP domain ofaggrecan G3, or both, thereby inhibiting activation of monocytes andother cells. In some embodiments, the agent comprises a soluble form ofthe PAMP receptor or DAMP receptor that binds to the PAMP domain offibronectin, the DAMP domain of fibronectin, or both, thereby inhibitingactivation of monocytes and other cells. In some embodiments, the agentinhibits production of proinflammatory cytokines, chemokines, proteases,or any combination thereof. In some embodiments, the agent inhibitsfibroblast cells, thereby inhibiting production of fibronectin,recruitment of other fibroblast cells, or a combination thereof. In someembodiments, the small molecule or polypeptide is identified using oneor more high-throughput screening methods. In some embodiments, thesmall molecule or polypeptide inhibits FAC formation, inhibitsactivation of monocytes, inhibits increased production of fibronectin,inhibits recruitment of fibroblast cells, binds to the DAMP domain offibronectin, binds to the DAMP domain of aggrecan G3, binds to the PAMPdomain of fibronectin, or binds to the PAMP domain of aggrecan G3. Insome embodiments, the small molecule or polypeptide inhibits FACformation by competitively binding to fibronectin or aggrecan. In someembodiments, the small molecule or polypeptide binds to the FAC complexresulting in dissociation or degradation of the FAC complex. In someembodiments, inhibiting the formation of the fibronectin-aggrecancomplex (FAC) comprises inhibiting of one or more steps in FACformation. In some embodiments, the one or more steps in FAC formationcomprise production of fibronectin in the ECM, production of proteasesand metalloproteases, production of inflammatory cytokines andchemokines, degradation of aggrecan in cartilage, and production ofaggrecan G3 domain fragment. In some embodiments, the conditioncomprises arthritis, inflammation, ligament injury, tendon injury, boneinjury, cartilage degeneration, cartilage injury an autoimmune disease,back pain, joint pain, joint degeneration, disc degeneration, spinedegeneration, bone degeneration, inflammation in joint or disc surgery,inflammation in joint or disc replacement, or any combination thereof.

In one aspect, provided herein is a composition comprising a variant A2Mpolypeptide, comprising a bait region, wherein the bait region of thevariant A2M polypeptide comprises a plurality of protease recognitionsites arranged in series. In some embodiments, the variant A2Mpolypeptide protein is a recombinant protein. In some embodiments, thevariant A2M polypeptide protein is produced in a host comprisingbacteria, yeast, fungi, insect, or mammalian cells, or a cell freesystem. In some embodiments, the variant A2M polypeptide protein ischaracterized by an enhanced nonspecific inhibition of serine proteases,threonine proteases, cysteine proteases, aspartate proteases,metalloproteases, glutamic acid proteases, or any combination thereof.In some embodiments, the variant A2M polypeptide protein furthercomprises PEG with abnormal glycosylation sites. In some embodiments,the variant A2M polypeptide protein has a longer half life than the halflife of a wild type A2M protein when disposed within a joint or spinedisc of a subject. In some embodiments, the plurality of proteaserecognition sites comprise one or more protease substrate bait regionsfrom one or more proteins other than A2M, one or more additionalprotease bait regions from A2M, one or more non-natural proteinsequences, or any combination thereof, wherein the modified A2M proteinis characterized by at least a 10% increase in protease inhibitoryeffectiveness compared to the protease inhibitory effectiveness of awild type A2M protein. In some embodiments, the non-natural proteinsequences comprise one or more protease recognition sites that canfunction as bait for proteases. In some embodiments, the one or moreprotease substrate bait regions comprise consensus sequences for serineproteases, threonine proteases, cysteine proteases, aspartate proteases,metalloproteinases, glutamic acid proteases, or any combination thereof.In some embodiments, the protease substrate bait regions comprise one ormore consensus sequences for one or more proteases from one or moreorganisms. In some embodiments, the one or more organisms compriseanimals, plants, bacteria, yeast, fish, reptiles, amphibians, or fungi.In some embodiments, one or more of the one or more protease substratebait regions from the one or more proteins other than A2M are the same.In some embodiments, one or more of the one or more protease substratebait regions from A2M are the same. In some embodiments, one or more ofthe one or more protease substrate bait regions from the one or morenon-natural protein sequences are the same. In some embodiments, one ormore of the one or more protease substrate bait regions from the one ormore proteins other than A2M or from the one or more non-natural proteinsequences comprise a suicide inhibitor; wherein the suicide inhibitor isoperable to covalently attach a protease to A2M. In some embodiments,one or more of the one or more protease substrate bait regions are fromdifferent species.

In one aspect, provided herein is a composition comprising an isolatedvariant A2M polypeptide, wherein the variant A2M polypeptide comprisesone or more non-natural bait regions, wherein the one or morenon-natural bait regions comprise one or more protease recognition sitesnot present in a wild-type A2M polypeptide. In some embodiments, themodified A2M polypeptide is characterized by at least a 10% enhancedinhibition of one or more proteases compared to a wild-type A2Minhibition of the one or more proteases. In some embodiments, theenhanced inhibition comprises enhanced nonspecific inhibition. In someembodiments, the enhanced inhibition comprises enhanced specificinhibition. In some embodiments, the protease comprises a serineprotease, threonine protease, cysteine protease, aspartate protease,metalloprotease, glutamic acid protease, or any combination thereof. Insome embodiments, the protease comprises MMP1 (Interstitialcollagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin 1), MMP7(Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11), Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP27 (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease, such as ADAMTS1, ADAMTS2, ADAMTS3,ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (METH-2),ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16,ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin; elastase;compliment factors; clotting factors; thrombin; plasmin; subtilisin;Neprilysin; Procollagen peptidase; Thermolysin; Pregnancy-associatedplasma protein A; Bone morphogenetic protein 1; Lysostaphin; Insulindegrading enzyme; ZMPSTE2; acetylcholinesterase; or a combinationthereof In some embodiments, the protease comprises ADAMTS4, ADAMTS 5,MMP13, or a combination thereof. In some embodiments, the modified A2Mpolypeptide is characterized by at least a 10% enhanced inhibition ofFAC formation compared to a wild-type A2M inhibition of FAC formation.In some embodiments, the one or more non-natural bait regions arederived from one or more proteins other than A2M. In some embodiments,the one or more proteins other than A2M are from a non-human organism.In some embodiments, the non-human organism comprises an animal, plant,bacterium, yeast, fish, reptile, amphibian, or fungi. In someembodiments, the one or more non-natural bait regions comprise SEQ IDNOs 5-66. In some embodiments, the variant A2M polypeptide comprises SEQID NO 4, or a fragment thereof. In some embodiments, the one or morenon-natural bait regions comprise SEQ ID NOs 5-66, or fragments thereof.In some embodiments, the wild-type A2M polypeptide comprises SEQ ID NO3, or a fragment thereof. In some embodiments, one or more of the one ormore non-natural bait regions comprise a suicide inhibitor; wherein thesuicide inhibitor is operable to covalently attach a protease to thevariant A2M polypeptide. In some embodiments, the one or more proteaserecognition sites comprise 2 or more copies of the one or more proteaserecognition sequences. In some embodiments, the one or more non-naturalbait regions comprise 2 or more copies of the one or more non-naturalbait regions. In some embodiments, the variant A2M polypeptide comprisesa wild-type A2M bait region sequence. In some embodiments, the variantA2M polypeptide is a recombinant polypeptide. In some embodiments, theone or more protease recognition sites comprise a consensus sequence fora protease. In some embodiments, the variant A2M polypeptide comprisesone or more modified glycosylation sites. In some embodiments, the oneor more modified glycosylation sites are functionalized with PEG. Insome embodiments, the variant A2M polypeptide has at least a 10% longerhalf life than the half life of a wild type A2M polypeptide whendisposed within a subject.

In one aspect, provided herein is a method of treating a subject withone or more conditions, comprising administering to the subject aneffective amount of any composition provided herein, a wild-type A2Mprotein, A2M variant, or a combination thereof. In some embodiments,nonspecific inhibition of one or more proteases in the subject,inhibition Aggrecan G3 fragment formation, inhibition FAC formation, ora combination thereof, is increased. In some embodiments, the rate ofdegeneration of tissue, cartilage and discs, synovial inflammation, or acombination thereof, is decreased in the subject. In some embodiments,treating results in a reduction in severity, occurrence, rate ofprogression, or a combination thereof, of the one or more conditions. Insome embodiments, any of the methods provided herein further compriseadministering one or more additional carriers or drugs. In someembodiments, the one or more additional carriers or drugs comprisehydrogels, hyaluronic acid preparations, polymer microspheres,corticosteroids, microparticles, chitosan, local anaesthetics, growthfactors, cytokines, protease inhibitors, steroids, hyaluranic Acid (HA),or other biologically active autogenous or endogenous mediators. In someembodiments, the one or more conditions are treatable with anycomposition provided herein. In some embodiments, the one or moreconditions comprise cancer, degenerative diseases, traumatic diseases,and/or inflammatory diseases, whose pathogenesis includes the activityof proteases. In some embodiments, the cancer, degenerative diseases,traumatic diseases, and/or inflammatory diseases whose pathogenesisincludes the activity of proteases comprises osteoarthritis,inflammatory arthritides, chondrosis, chondral injuries, enthesopathies,tendinopathies, ligamentous injuries, degenerative diseases of the bone,cartilage, tendons, and ligaments, post-operative conditions and woundhealing, and other musculoskeletal diseases. In some embodiments, theone or more conditions comprise cancer, arthritis, inflammation,ligament injury, tendon injury, bone injury, cartilage degeneration,cartilage injury, an autoimmune disease, back pain, joint pain, jointdegeneration, disc degeneration, spine degeneration, bone degeneration,or any combination thereof. In some embodiments, inflammation comprisesjoint or disc inflammation caused by surgery, joint or disc inflammationcaused by a joint or disc replacement, or a combination thereof. In someembodiments, the subject is a human, pig, mouse, rat, rabbit, cat, dog,monkey, frog, horse or goat. In some embodiments, the subject has beenpreviously diagnosed with the one or more conditions. In someembodiments, the composition is administered into an anatomic siterelevant to the host pathology. In some embodiments, the administrationcomprises injection with a hollow-lumen device or flexible cathetercombinations. In some embodiments, the hollow-lumen device comprises aneedle, syringe, or combination thereof. In some embodiments, theadministration occurs during a surgical procedure.

In one aspect, provided herein is a composition comprising an isolatedvariant A2M polynucleotide, wherein the variant A2M polynucleotideencodes for one or more non-natural bait regions, wherein the one ormore non-natural bait regions comprise one or more protease recognitionsites not present in a wild-type A2M polypeptide. In some embodiments,the non-natural bait regions comprise a sequence with at least 60%identity to SEQ ID NOs 5-66, or fragments thereof. In some embodiments,the variant A2M polynucleotide comprises at least 90% identity to SEQ IDNO 2, or a fragment thereof. In some embodiments, the wild-type A2Mpolynucleotide comprises SEQ ID NO 1, or a fragment thereof. In someembodiments, the variant A2M polynucleotide is within an expressionvector.

In one aspect, provided herein is a method for determining the enhancedinhibition of a protease by a variant A2M polypeptide comprising: (a)providing a variant A2M polypeptide comprising a sequence of one or moreof SEQ ID NOs 5-66; (b) contacting the variant A2M polypeptide with theprotease and a substrate cleaved by the protease; (c) contacting awild-type A2M polypeptide with the protease and the substrate cleaved bythe protease; and (d) comparing the amount of cleavage of the substratefrom step (b) to the amount of cleavage of the substrate from step (c),thereby determining the enhanced inhibition of the protease by thevariant A2M polypeptide.

In one aspect, provided herein is a method for making a variant A2Mpolynucleotide comprising: (a) providing a vector containing a variantA2M polynucleotide comprising a sequence of SEQ ID NO 2; (b) digestingthe vector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; (c) ligating one end of the oneor more polynucleotides encoding one or more of the non-natural baitregions of SEQ ID NOs 5-66 to one end of the linear vector; and (d)ligating the other end of the one or more polynucleotides encoding oneor more of the non-natural bait regions of SEQ ID NOs 5-66 to the otherend of the linear vector, thereby forming a vector containing a variantA2M polynucleotide comprising the non-natural bait regions of SEQ ID NOs5-66.

In one aspect, provided herein is a composition comprising A2M, whereinthe composition is obtainable by any method provided herein. In someembodiments, the composition is for autologous administration.

In one aspect, provided herein is a composition comprising A2M for usein therapy wherein the composition is (a) any liquid compositionprovided herein (b) a composition obtainable by any method providedherein, or (c) any variant A2M composition provided herein In someembodiments, the composition is for use in autologous therapy or for usein non-autologous therapy. In some embodiments, the composition is foruse in the treatment of cancer, arthritis, inflammation, ligamentinjury, tendon injury, bone injury, cartilage degeneration, cartilageinjury, an autoimmune disease, back pain, joint pain, jointdegeneration, disc degeneration, spine degeneration, bone degeneration,or any combination thereof wherein inflammation comprises joint or discinflammation caused by surgery, joint or disc inflammation caused by ajoint or disc replacement, or a combination thereof.

In one aspect, provided herein is a use of (a) any liquid compositionprovided herein (b) a composition obtainable by any method providedherein, or (c) any variant A2M composition provided herein, for themanufacture of a medicament for use in therapy. In some embodiments, themedicament is for use in autologous therapy or for use in non-autologoustherapy. In some embodiments, the medicament is for use in the treatmentof cancer, arthritis, inflammation, ligament injury, tendon injury, boneinjury, cartilage degeneration, cartilage injury, an autoimmune disease,back pain, joint pain, joint degeneration, disc degeneration, spinedegeneration, bone degeneration, or any combination thereof whereininflammation comprises joint or disc inflammation caused by surgery,joint or disc inflammation caused by a joint or disc replacement, or acombination thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.In the event of a conflict between a term herein and a term incorporatedby reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features are set forth with particularity in the appendedclaims. A better understanding of the features and advantages will beobtained by reference to the following detailed description that setsforth illustrative embodiments, in which the principles of devices,methods, and compositions are utilized, and the accompanying drawings ofwhich:

FIG. 1 depicts a schematic of the steps and signaling pathwaysassociated with formation of a fibronectin-aggrecan complex (FAC) andthe FAC-induced activation of Damage-Associated-Molecular Pattern (DAMP)receptor signaling in cells. The combination of the two processescreates a cyclic process that continually degrades cartilage.

FIG. 2 depicts FAC formation using fibronectin to form a complex withpurified full length Aggrecan or recombinant G3 Aggrecan. Both Aggrecanand the G3 domain bind fibronectin to form FAC.

FIG. 3 depicts a flow chart of the steps for construct or proteinexpression.

FIG. 4 depicts the A2M structure and various domains of A2M.

FIG. 5A depicts a graph demonstrating treatment of Bovine CartilageExplants (BCE) with leukocyte-rich Platelet Rich Plasma (LR-PRP), whichinduces cartilage catabolism, and treatment with purified A2M to inhibitcartilage degradation.

FIG. 5B depicts a graph demonstrating treatment of Bovine CartilageExplants (BCE) with APIC-PRP, blood, or leukocyte-rich Platelet RichPlasma (LR-PRP) from the same patient. LR-PRP, but not blood, inducescartilage catabolism. Treatment of BCE with APIC-PRP inhibits cartilagedegradation below endogenous levels.

FIG. 5C depicts a graph demonstrating leukocyte-rich Platelet RichPlasma (LR-PRP) induces cartilage catabolism in a Bovine CartilageExplant (BCE) model. Treatment with APIC-PRP inhibits the cartilagedegradation induced by treatment with LR-PRP.

FIG. 6A depicts a graph showing Bovine Cartilage Explants (BCE) treatedwith pro-inflammatory cytokines TNF-α and IL-1β to induce cartilagecatabolism. Cartilage catabolism with each cytokines separately isdemonstrated by the release of sulfated Glycosaminoglycans (sGAG) intothe culture media. Treatment with APIC-PRP efficiently inhibitscartilage catabolism by each pro-inflammatory cytokine separately.

FIG. 6B depicts a graph showing Bovine Cartilage Explants (BCE) treatedwith the combination of pro-inflammatory cytokines TNF-α and IL-1β toinduce cartilage catabolism. Treatment with APIC-PRP efficientlyinhibited cartilage catabolism by the combination of pro-inflammatorycytokines in a dose dependent manner.

FIG. 7A depicts the sulfated glycosaminoglycan (sGAG) released uponcartilage catabolism in a BCE model with and without treatment ofADAMTS-5 and treatment with or without a serial dilution of purified A2M(top). Western Blots of the samples (bottom) demonstrate ADAMTS-5degradation of cartilage produced an Aggrecan G3 fragment and highermolecular weight Aggrecan fragments, which were inhibited by treatmentwith A2M in a dose dependent manner. Values above the columns indicatethe concentration of A2M (μg/ml) needed to inhibit ADATMS-5. An 85 kDanon-specific band is also visible, which was apparent in media-onlycontrols (data not shown).

FIG. 7B depicts the sulfated glycosaminoglycan (sGAG) released uponcartilage catabolism in a BCE model with and without treatment ofADAMTS-4 and treatment with or without a serial dilution of purified A2M(top). Western Blot analysis with α-Aggrecan G3 antibody (bottom) of thesamples demonstrates ADAMTS-4 degradation of cartilage produced highmolecular weight Aggrecan C-terminal fragments containing the G3 domain.Cartilage catabolism is inhibited by A2M in a dose dependent manner andreduces the release of cartilage aggrecan fragments. An 85 kDanon-specific band is also visible, which was apparent in media-onlycontrols (data not shown).

FIG. 8A depicts a graph demonstrating the sulfated glycosaminoglycan(sGAG) released upon cartilage catabolism in a BCE model with andwithout treatment of MMP-7 and MMP-12. Treatment with purified A2Minhibited the MMP-induced cartilage catabolism.

FIG. 8B depicts a stained SDS-PAGE gel of samples produced in FIG. 9A.The MMP-7- or MMP-12-induced degradation of cartilage, and theproduction of cartilage protein fragments visible in the gel, wasinhibited with addition of purified A2M.

FIG. 8C depicts a Western Blot with α-Aggrecan G3 antibody using the gelfrom FIG. 8B and the samples from FIG. 8A. The degradation of cartilageby MMP-7 or MMP-12 produces an Aggrecan G3 fragment at −30 kDa which canbe inhibited with addition of purified A2M.

FIG. 9 depicts the results of an ELISA test that recognizes complexes ofFibronectin and Aggrecan G3 (FACT, Fibronectin Aggrecan Complex Test).Culture media from BCE treated with or without the listed proteases inthe presence or absence of A2M were incubated with Synovial Fluid (SF)spiked with free Fibronectin and tested on the FACT assay. In each casewhere degradation of cartilage led to Aggrecan fragments the result wasformation of additional Fibronectin Aggrecan Complexes above the SFbackground control. Treatment with A2M, however, which preventedcartilage catabolism, subsequently preventing FAC formation.

FIG. 10 depicts two bar graphs demonstrating the ability of APIC(Retentate from the 500 kDa filter) and the Filtrate to preventcartilage degradation. Cartilage catabolism was induced in the BCE modelwith ADAMTS-5, which could be inhibited with serial dilution of APIC(left, Retentate), but not the Filtrate which is devoid of A2M (right,Filtrate). The numbers above the columns represent the percentage ofAPIC (v/v) or filtrate in the culture media. The inhibitory potential in5% of Filtrate is equivalent to 0.01% of APIC; thus the process ofproducing APIC concentrates >99% of the chondroprotective effects ofblood.

FIG. 11 is a bar graph depicting the effects of treatment of THP-1monocytes with autologous APIC for two days in culture. No activation ofthe monocytes was observed through monitoring with a panel of cytokines,chemokines, and growth factors (Left to right: IL-1β, IL-1 receptoragonist (IL-1ra), IL-6, IFN-γ, IP-10, MCP-10, MIP-1β, PDGF-ββ, RANTES,TNF-α, and VEGF).

FIG. 12 depicts macroscopic images of rabbit knees 6 weeks after ACL-Tsurgery and treatment with saline or APIC cell free. Sham surgerieswithout ACL-T were performed as a control.

FIG. 13A depicts a graph of macroscopic evaluation for the experimentsshown in FIG. 12. The values shown are the average of the macroscopicevaluation of 6 rabbits.

FIG. 13B depicts a graph of macroscopic evaluation, showing an inversecorrelation of A2M in APIC cell free treatment and cartilage degradationfor the experiments shown in FIG. 12.

FIG. 14 depicts graphs of histopathology evaluation of the rabbit kneesfrom experiments depicted in FIGS. 12 and 13 including structure,chondrocyte density, Safarin-O staining, and cluster formationevaluations; and shows an inverse correlation between A2M concentrationin each rabbit's APIC and the scoring criteria. One outlying rabbit isexcluded from calculations in the line but is included in the figures.

FIG. 15 is a depiction of a pseudocolored stain-free SDS-PAGE gel of arepresentative purification of tagged wild-type A2M and the fourselected variable bait region A2M proteins. The theoretical molecularweight of a monomer of wild-type A2M is 163 KDa, not includingglycosylation. The blurry band above 250 KDa is comprised of dimeric A2Mthat is not thoroughly reduced during sample preparation or covalentlybound dimer through amino acid modification mechanisms.

FIG. 16 is a depiction of a pseudocolored stain-free SDS-PAGE gel (top)and Western blot (bottom) of a representative screening assay forinhibition of ADAMTS-5 cleavage of aggrecan IGD domain (IGD fragment) bywild-type (WT) and bait region substituted A2M. The negative control isIGD fragment protein alone; the positive control is IGD fragment plusADAMTS-5. ADAMTS-5, Wild-type and variant A2M were each kept at 50 nM,and the A2M and ADAMTS-5 were pre-mixed for 10 min. before addition ofIGD fragment. The primary antibody for the Western blot was ananti-Aggrecan G1-IGD-G2 polyclonal antibody (R&D).

FIG. 17 is a graph depicting a comparison of the relative inhibitorycharacteristics of the four chosen variants vs. various MMPs andADAMTS-4 and -5 as determined by the two IGD screening experiments. Ineach case the unit for the y-axis is multiples of the wild-typeinhibition of each protease.

FIG. 18A depicts the raw data (left) and calculated slope (right) ofdigestion of FTC-casein by bovine trypsin in the presence of taggedwild-type A2M (WT) or the four chosen A2M variants. The samples withoutthe “−D” are prepared with a 1:1 molar ratio of A2M:protease. Those withthe “−D” are prepared at a 0.5:1 ratio of A2M:protease.

FIG. 18B depicts the raw data (left) and calculated slope (right) ofdigestion of FTC-casein by chymotrypsin in the presence of taggedwild-type A2M (WT) or the four chosen A2M variants. The samples withoutthe “−D” are prepared with a 1:1 molar ratio of A2M:protease. Those withthe “−D” are prepared at a 0.5:1 ratio of A2M:protease.

FIG. 19 depicts a western blot analysis of a cleavage assay using IGDfragment as a substrate in the presence of the MMP3.

FIG. 20 depicts a chart of the inhibition of IGD fragment proteolysis bythe indicated variants as a percentage of wild-type A2M (top) and thesequences of the bait sequences corresponding to the indicated A2Mvariants (bottom).

FIG. 21 depicts Western blots showing the control blot of degraded andnon-degraded forms of A2M as a function of the known amount of proteinindicated (top) and the cleavage of various A2M polypeptides over timein the presence of a protease (bottom). The control blot can be used toquantify the amount of cleaved A2M, which is directly proportional tothe rate of protease inhibition.

FIG. 22 depicts the protective effect of the A2M wild type vs. some ofthe variants of the digestion of IGD domain from a mixture of proteases.10 nM of each MMP1, MMP3, MMP7, MMP13, ADAMTS4 and ADAMTS5 were mixedand used to digest IGD in the presence or absence of A2M wild type andA2M variants.

FIG. 23 depicts a Vector Map of pJ608 mammalian expression vector. TheORF sequence coding for wild-type and variant A2M is cloned in betweenthe Kpn1 and BamH1 restriction sites.

FIG. 24 depicts a schematic of a system as described herein.

FIG. 25 a picture of the concentration kit/tray of a system describedherein showing one filter, a concentration bag and the filtrate bag.

FIG. 26 depicts a schematic of the components of a concentration bag ofa system described herein.

FIG. 27 depicts a schematic of a system process overview.

FIG. 28 depicts the components of a system described herein showing acentrifuge and a one filter system.

FIG. 29 depicts two different types of custom centrifuge tubes that canbe used in the systems described herein.

FIG. 30 depicts a custom centrifuge used in the systems describedherein.

FIG. 31 depicts a schematic of the components of a concentration bag ofa cell free concentration system as described herein where two filtersare utilized and no centrifugation step.

FIG. 32 depicts a schematic of a cell free concentration system asdescribed herein with concentration component utilizing two filters.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are compositions, methods, kits and systems for thedetection, diagnosis, and treatment of inflammation, pain in the spineor joint, and degradation of extracellular matrix.

The details of one or more inventive embodiments are set forth in theaccompanying drawings, the claims, and in the description herein. Otherfeatures, objects, and advantages of inventive embodiments disclosed andcontemplated herein will be apparent from the description and drawings,and from the claims. As used herein, unless otherwise indicated, thearticle “a” means one or more unless explicitly otherwise provided for.As used herein, unless otherwise indicated, terms such as “contain,”“containing,” “include,” “including,” and the like mean “comprising.” Asused herein, unless otherwise indicated, the term “or” can beconjunctive or disjunctive. As used herein, unless otherwise indicated,any embodiment can be combined with any other embodiment. As usedherein, unless otherwise indicated, some inventive embodiments hereincontemplate numerical ranges. When ranges are present, the rangesinclude the range endpoints. Additionally, every subrange and valuewithin the range is present as if explicitly written out.

Definitions

The term “substantially non-immunogenic” or “substantiallynon-antigenic” means that the composition being administered to asubject does not elicit an immune response to the composition.

A “subject” refers to a donor, recipient or host of the composition ofthe present invention. In some embodiments, the donor and the recipientare the same. In some embodiments the subject is a human subject.

A “proteoglycan” refers to a special class of proteins that are heavilyglycosylated. A proteoglycan is made up of a core protein with numerouscovalently attached high sulphated glycosaminoglycan chain(s).Non-limiting example of extracellular matrix proteoglycans includeaggrecan and certain collagens, such as collagen IX.

A “glycosaminoglycan” or “GAG” as used herein refers to a longunbranched polysaccharide molecules found on the cell surface or withinthe extracellular matrix. Non-limiting examples of glycosaminoglycaninclude heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate,heparan sulfate, keratan sulfate, hyaluronic acid, hexuronylhexosaminoglycan sulfate, and inositol hexasulfate.

The term “non-autologous” refers to tissue or cells which originate froma donor other than the recipient. Non-autologous can refer to, forexample, allogeneic or xenogeneic. The term “autologous” as in anautologous composition, refers to a composition in which the donor andrecipient is the same individual. Likewise, “allogeneic” refers to adonor and a recipient of the same species; “syngeneic” refers to a donorand recipient with identical genetic make-up (e.g. identical twins orautogeneic) and “xenogeneic” refers to donor and recipient of differentspecies.

The term “variant” (or “analog”) refers to any molecule differing fromthe naturally occurring molecule.

The term “variant polynucleotide” (or “analog”) refers to anypolynucleotide differing from the naturally occurring polynucleotide.For example, “variant A2M polynucleotide” refers to any A2Mpolynucleotide differing from naturally occurring A2M polynucleotides. Avariant A2M polynucleotide includes a polynucleotide sequence differentfrom the wild-type A2M polynucleotide sequence (SEQ ID NO: 1). Variantpolynucleotides can be characterized by nucleic acid insertions,deletions, and substitutions, created using, for example, recombinantDNA techniques. A variant A2M polynucleotide preferably includes amutation, insertion, deletion, or a combination thereof, in the baitregion of a wild-type A2M polynucleotide sequence. As used herein, whenreferring to polypeptides, the “bait region” includes the region of anA2M polynucleotide that encodes the region of the A2M polypeptide thatbinds to proteases, for example, regions that contain proteaserecognition sites. A variant A2M polynucleotide includes an “A2Macceptor sequence” (SEQ ID NO: 2) which includes a polynucleotidesequence of A2M with point mutations that can aid in creating variantA2M polynucleotides by recombinant DNA techniques, for example, bycreating restriction enzyme cloning sites to aid in inserting variouspolynucleotide sequences encoding the variant bait regions. Bait regionsinclude SEQ ID NOs: 5-66 and sequences substantially similar to SEQ IDNOs: 5-66.

The term “variant polypeptide” refers to any polypeptide differing fromthe naturally occurring polypeptide. For example, “variant A2Mpolypeptide” refers to any A2M polypeptide differing from naturallyoccurring A2M polypeptides. Variant polypeptides can be characterized byamino acid insertions, deletions, and substitutions, created using, forexample, recombinant DNA techniques. A variant A2M polypeptide includesa polypeptide sequence different from the wild-type A2M polypeptidesequence. A variant A2M polypeptide preferably includes a mutation,insertion, deletion, or a combination thereof, in the bait region of awild-type A2M protein. When referring to polypeptides, the “bait region”includes the region of an A2M polypeptide that binds to proteases, forexample, a stretch of amino acids that contains one or more proteaserecognition sites. A variant A2M polypeptide includes a polypeptide (SEQID NO: 3) encoded by an A2M acceptor sequence (SEQ ID NO: 2). A “variantA2M polypeptide” can have at least one amino acid sequence alteration inthe bait region as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be, for example, a substitution, a deletion, or an insertion of oneor more amino acids. A variant A2M polypeptide can have any combinationof amino acid substitutions, deletions or insertions.

Guidance in determining which amino acid residues may be replaced, addedor deleted without abolishing activities of interest, may be found bycomparing the sequence of the particular polypeptide with that ofhomologous peptides and minimizing the number of amino acid sequencechanges made in regions of high homology (conserved regions) or byreplacing amino acids with consensus sequence. Alternatively,recombinant variants encoding these same or similar polypeptides may besynthesized or selected by making use of the “redundancy” in the geneticcode. Various codon substitutions, such as the silent changes whichproduce various restriction sites, may be introduced to optimize cloninginto a plasmid or viral vector or expression in a particular prokaryoticor eukaryotic system. Mutations in the polynucleotide sequence may bereflected in the polypeptide or domains of other peptides added to thepolypeptide to modify the properties of any part of the polypeptide, tochange characteristics such as inhibition of proteases, ligand-bindingaffinities, interchain affinities, or degradation/turnover rate. Variantnucleotides can also be used to generate polypeptides that are bettersuited for expression, scale up and the like in the host cells chosenfor expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

An amino acid “substitution” includes replacing one amino acid withanother amino acid having similar structural and/or chemical properties,for example, conservative amino acid replacements. “Conservative” aminoacid substitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, the amphipathicnature of the residues involved, or a combination thereof. Nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. Polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. Positively charged (basic) amino acidsinclude arginine, lysine, and histidine. Negatively charged (acidic)amino acids include aspartic acid and glutamic acid. “Insertions” or“deletions” are preferably in the range of about 1 to 50 amino acids,more preferably 1 to 30 amino acids. The variation allowed can beexperimentally determined by inserting, deleting, or substituting aminoacids in a polypeptide using recombinant DNA techniques and assaying theresulting recombinant variants for activity, for example, proteaseinhibition activity.

The terms “purified” or “substantially purified” as used herein denotesthat the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, for example,polynucleotides, proteins, and the like. The polynucleotide orpolypeptide can be purified such that it constitutes at least 95% byweight, for example, at least 99% by weight, of the indicated biologicalmacromolecules present. Water, buffers, and other small molecules with amolecular weight of less than 1000 Daltons, can be present in anyamount. The term “isolated” as used herein refers to a polynucleotide orpolypeptide separated from at least one other component present with thepolynucleotide or polypeptide in its natural source. In someembodiments, the polynucleotide or polypeptide can be found in thepresence of only a solvent, buffer, ion, or other components normallypresent in a solution of the same. The terms “isolated” and “purified”do not encompass polynucleotides or polypeptides present in theirnatural source.

As used herein, “recombinant polypeptides” include polypeptides orproteins derived from recombinant expression systems, for example,microbial, insect, or mammalian expression systems. Polypeptides orproteins expressed in most bacterial cultures will be free ofglycosylation modifications; polypeptides or proteins expressed in yeastcan have a glycosylation pattern in general different from thoseexpressed in mammalian cells.

The term “expression vector” refers to a plasmid or phage or virus orvector, for expressing a polypeptide from a DNA or RNA sequence. Anexpression vector can include a transcriptional unit comprising anassembly of a genetic element or elements having a regulatory role ingene expression, for example, promoters or enhancers, a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems can include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an amino terminal methionine residue. This residue may ormay not be subsequently cleaved from the expressed recombinant proteinto provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems can be used to express heterologouspolypeptides or proteins upon induction of the regulatory elementslinked to the DNA segment or synthetic gene to be expressed. This termincludes host cells which have stably integrated a recombinant geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers. Recombinant expression systems can beused to express polypeptides or proteins endogenous to the cell uponinduction of the regulatory elements linked to the endogenous DNAsegment or gene to be expressed. The cells can be prokaryotic oreukaryotic.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly,for example soluble proteins, or partially, for example receptors, fromthe cell in which they are expressed. “Secreted” proteins also includeproteins transported across the membrane of the endoplasmic reticulum.“Secreted” proteins also include proteins containing non-typical signalsequences.

Where desired, an expression vector may be designed to contain a “signalsequence” which will direct the polypeptide through the membrane of acell. A signal sequence can be naturally present on the polypeptidesdescribed herein or provided from heterologous protein sources.

As used herein, “substantially equivalent” or “substantially similar”can refer both to nucleotide and amino acid sequences, for example avariant sequence, that varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which does notresult in an adverse functional dissimilarity between the reference andsubject sequences. Typically, such a substantially equivalent sequencevaries from one of those listed herein by no more than about 35%. Forexample, the number of individual residue substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.35 or less.A substantially equivalent sequence includes sequences with 65% sequenceidentity to the reference sequence. A substantially equivalent sequenceof the invention can vary from a reference sequence by no more than 30%(70% sequence identity), no more than 25% (75% sequence identity), nomore than 20% (80% sequence identity), no more than 10% (90% sequenceidentity), or no more that 5% (95% sequence identity). Substantiallyequivalent amino acid sequences according to the invention preferablyhave at least 80% sequence identity with a reference amino acidsequence, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity. Substantially equivalentpolynucleotide sequences of the invention can have lower percentsequence identities, taking into account, for example, the redundancy ordegeneracy of the genetic code. Preferably, the polynucleotide sequencehas at least about 65%, at least about 75%, at least about 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. Sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. Identity between sequences can be determinedby methods known in the art, such as by alignment of the sequences orvarying hybridization conditions.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate spinalpain in a subject in need thereof.

By “degenerate variant” can be intended nucleotide fragments whichdiffer from a nucleic acid fragment of the present invention (e.g., anORF) by nucleotide sequence but, due to the degeneracy of the geneticcode, encode an identical polypeptide sequence.

The terms polypeptide, peptide, and protein can be used interchangeablyand can refer to a polymer of amino acid residues or a variant thereof.Amino acid polymers can have one or more amino acid residues and can bean artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers, thosecontaining modified residues, and non-naturally occurring amino acidpolymers. A variant polypeptide can have at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be, for example, a substitution, a deletion, or an insertion of oneor more amino acids. A variant polypeptide can have any combination ofamino acid substitutions, deletions or insertions. An amino acidsequence alteration can be formed by altering the nucleotide sequencefrom which it is derived, such as a mutation, for example, a frameshiftmutation, nonsense mutation, missense mutation, neutral mutation, orsilent mutation. For example, sequence differences, when compared to awild-type nucleotide sequence, can include the insertion or deletion ofa single nucleotide, or of more than one nucleotide, resulting in aframe shift; the change of at least one nucleotide, resulting in achange in the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of a reading frame; duplication ofall or a part of a sequence; transposition; or a rearrangement of anucleotide sequence. Such sequence changes can alter the polypeptideencoded by the nucleic acid, for example, if the change in the nucleicacid sequence causes a frame shift, the frame shift can result in achange in the encoded amino acids, and/or can result in the generationof a premature stop codon, causing generation of a truncatedpolypeptide.

The term “fragment” can refer to any subset of the polypeptide that canbe a shorter polypeptide of the full length protein. Fragments of A2Mcan include 20, 30, 40, 50 or more amino acids from A2M that can bedetected with anti-A2M antibodies. Other fragments of A2M includevarious domains of A2M and combinations thereof.

“Platelet-rich plasma” (“PRP”) can refer to blood plasma that has beenenriched with platelets.

Compositions for Autologous Treatment of Pain and Inflammation

An autologous composition of the present disclosure can comprisealpha-2-macroglobulin (A2M) and can be used to treat a subject with acondition. The A2M can be from a biological sample, such as from a humansubject; or can be any fragment thereof. In preferred embodiments theautologous compositions of the present invention are substantiallynon-immunogenic, namely do not elicit an immune response.

An autologous composition can comprise an elevated concentration of A2Mcompared to the concentration of A2M found in a biological sample, suchas the blood of normal subjects, or the blood from a subject in need ofautologous treatment with the autologous composition. The concentrationof A2M in an autologous composition can be at least about 1.1 timeshigher than the concentration of A2M found in a biological sample, suchas blood, of normal subjects. For example, the concentration of A2M canbe at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 times higher than the concentration of A2M foundin a biological sample, such as blood, of normal subjects, or the bloodfrom a subject in need of autologous treatment with the autologouscomposition. For example, the concentration of A2M can be at least about2 times higher than the concentration of A2M found in the biologicalsample.

In some embodiments, an autologous composition can further comprise areduced concentration of components other than A2M compared to thenormal concentration of the other components. An autologous compositioncan comprise a reduced concentration of other components isolated from abiological sample compared to the normal concentration of the othercomponents in the biological sample. The concentration of othercomponents can be at least about 10% less than the concentration of theother components normally found in a biological sample. For example, theconcentration of other components can be at least about 11%, 12%, 13%,14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%,800%, 900%, or 1000% less than the concentration of the other componentsnormally found in a biological sample, such as an endogenousconcentration of the other components in a biological sample. Forexample, the concentration of other components can be at least about 20%less than the concentration of the other components normally found in abiological sample. The concentration of other components can be at leastabout 0.1 times less than the concentration of the other componentsnormally found in a biological sample. For example, the concentration ofother components can be at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times less than the concentration of theother components normally found in a biological sample. For example, theconcentration of other components can be at least about 2 times lessthan the concentration of the other components normally found in abiological sample.

In some embodiments, an autologous composition can comprise an elevatedconcentration of one or more proteins with a molecular weight higherthan 100 kDa. The concentration of one or more proteins with a molecularweight higher than 100 kDa can be at least about 1.1 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 100 kDa found in a normal biological sample, such as bloodfrom a subject. The concentration of one or more proteins with amolecular weight higher than 100 kDa found in a normal biological samplecan be the concentration of the endogenous level of the one or moreproteins with a molecular weight higher than 100 kDa in the biologicalsample, such as a normal or control biological sample. For example, theconcentration of one or more proteins with molecular weight higher than100 kDa can be at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 times higher than the concentration of the one or more proteinswith molecular weight higher than 100 kDa found in a normal biologicalsample or the endogenous concentration in a normal biological sample.For example, the concentration of one or more proteins with molecularweight higher than 100 kDa can be at least about 1.5 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 100 kDa found in a normal biological sample or theendogenous concentration in a normal biological sample.

Proteins with a molecular weight higher than 100 kDa can include, butare not limited to, immunoglobulin G, immunoglobulin M, fibronectin,fibrinogen and other proteins. Proteins with a molecular weight lessthan about 100 kDa can comprise cytokines, chemokines, proteases,pro-proteases, enzymes, pro-enzymes, immune-modulators and otherproteins known in the art with a molecular weight of less than 100 kDa.

In some embodiments, an autologous composition can comprise an elevatedconcentration of one or more proteins with a molecular weight higherthan 500 kDa. The concentration of one or more proteins with a molecularweight higher than 500 kDa can be at least about 1.1 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 500 kDa found in a normal biological sample, such as bloodfrom a subject. The concentration of one or more proteins with amolecular weight higher than 500 kDa found in a normal biological samplecan be the concentration of the endogenous level of the one or moreproteins with a molecular weight higher than 500 kDa in a biologicalsample, such as a normal or control biological sample. For example, theconcentration of one or more proteins with molecular weight higher than100 kDa can be at least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 times higher than the concentration of the one or more proteinswith molecular weight higher than 500 kDa found in a normal biologicalsample or the endogenous concentration in a normal biological sample, orthe blood from a subject in need of autologous treatment with theautologous composition. For example, the concentration of one or moreproteins with molecular weight higher than 500 kDa can be at least about1.5 times higher than the concentration of the one or more proteins withmolecular weight higher than 100 kDa found in a normal biological sampleor the endogenous concentration in a normal biological sample. Proteinswith a molecular weight higher than 500 kDa include, but are not limitedto, IgM and Complement Component C4 binding proteins. Proteins with amolecular weight less than about 500 kDa can comprise cytokines,chemokines, proteases, pro-proteases, enzymes, pro-enzymes,immune-modulators and other proteins with a molecular weight of lessthan 500 kDa.

In some embodiments, proteins with a molecular weight between about 300kDa and 500 kDa, such as fibronectin, fibrinogen, and fibrin monomers orpolymers may be partially concentrated using the methods describedherein. In some embodiments, an autologous composition can comprise anelevated concentration of one or more proteins with a molecular weighthigher between about 300 kDa and 500 kDa. In some embodiments, theconcentration of one or more proteins with a molecular weight higherthan between about 300 kDa and 500 kDa can be at least about 1.1 timeshigher than the concentration of the one or more proteins with molecularweight between about 300 kDa and 500 kDa found in a normal biologicalsample, such as blood from a subject. The concentration of one or moreproteins with a molecular weight between about 300 kDa and 500 kDa foundin a normal biological sample can be the concentration of the endogenouslevel of the one or more proteins with a molecular weight between about300 kDa and 500 kDa in a biological sample, such as a normal or controlbiological sample. For example, the concentration of one or moreproteins with molecular weight between about 300 kDa and 500 kDa can beat least about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times higher than the concentration of the one or more proteins withmolecular weight between about 300 kDa and 500 kDa found in a normalbiological sample or the endogenous concentration in a normal biologicalsample.

An autologous composition can comprise an elevated concentration of A2Mcompared to the concentration of A2M found in a biological sample and areduced concentration of components other than A2M compared to thenormal concentration of the other components found in a biologicalsample. The concentration of A2M in an autologous composition can be atleast about 1.5 times higher than the concentration of A2M found in abiological sample and the concentration of other components other thanA2M can be at least about 10% less than the concentration of the othercomponents normally found in a biological sample. For example, theconcentration of A2M can be at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000 times higher than the concentration of A2M found in a biologicalsample and the concentration of components other than A2M can be atleast about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000times less than the concentration of the other components normally foundin a biological sample. For example, the concentration of theconcentration of A2M can be at least about 2 times higher than theconcentration of A2M found in a biological sample and the concentrationof components other than A2M can be at least about 2 times less than theconcentration of the other components normally found in a biologicalsample. As another example, the concentration of A2M can be at leastabout 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000 times higher than theconcentration of A2M found in a biological sample and the concentrationof other components can be at least about 11%, 12%, 13%, 14%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or1000% less than the concentration of the other components normally foundin a biological sample. For example, the concentration of A2M can be atleast about 2 times higher than the concentration of A2M found in abiological sample and the concentration of other components can be atleast about 20% less than the concentration of the other componentsnormally found in a biological sample.

In some embodiments, an autologous composition can comprise an elevatedconcentration of A2M compared to the concentration of A2M found in abiological sample and an elevated concentration of one or more proteinswith molecular weight higher than 100 kDa found in a biological sample.The concentration of A2M in an autologous composition can be at leastabout 1.5 times higher than the concentration of A2M found in abiological sample and the concentration of one or more proteins withmolecular weight higher than 100 kDa can be at least about 1.5 timeshigher than the concentration of the one or more proteins with molecularweight higher than 100 kDa found in a biological sample. For example,the concentration of the concentration of A2M can be at least about 1.6,1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 100 kDa can be at least about1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 times higher than the concentration ofthe one or more proteins with molecular weight higher than about 100 kDafound in a biological sample. For example, the concentration of A2M canbe at least about 2 times higher than the concentration of A2M found ina biological sample and the concentration of one or more proteins withmolecular weight higher than 100 kDa can be at least about 2 timeshigher than the concentration of the one or more proteins with molecularweight higher than about 100 kDa found in a biological sample. Proteinswith a molecular weight higher than about 100 kDa can be, for example,proteins with a molecular weight more than about 150 kDa, 200 kDa, 250kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa,1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa,1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa,1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, or more.

The concentration of A2M and other proteins with a molecular weighthigher than 100 kDa can be present at a concentration of at least about1.5 times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein. For example, the concentration of A2M and other proteins with amolecular weight higher than 100 kDa can be present at a concentrationof at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein. For example, theconcentration of A2M and other proteins with a molecular weight higherthan 100 kDa can be present at a concentration of at least about 1.5times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein.

The concentration of proteins with molecular weight less than about 100kDa can be less than about 10% of the concentrations of those proteinsin a biological sample when not retained by the one or more filtersusing the methods or systems described herein. For example, theconcentration of proteins with molecular weight less than about 100 kDacan be less than about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% less than theirconcentration in a biological sample when not retained by the one ormore filters using the methods or systems described herein. For example,the concentration of proteins with molecular weight less than about 100kDa can be less than about 20% less than their concentration in abiological sample when not retained by the one or more filters using themethods or systems described herein. Proteins with a molecular weightless than about 100 kDa can be, for example, proteins with a molecularweight less than about 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa,65 kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa,20 kDa, 15 kDa, 10 kDa, 5 kDa, or less.

The concentration of A2M and other proteins with a molecular weighthigher than 100 kDa can be present at a concentration of at least about1.5 times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein and the concentration of proteins with molecular weight less thanabout 100 kDa can be less than about 10% of the concentrations of thoseproteins in a biological sample when not retained by the one or morefilters using the methods or systems described herein. For example, theconcentration of A2M and other proteins with a molecular weight higherthan 100 kDa can be present at a concentration of at least about 1.6,1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than their concentration in abiological sample after retention by one or more filters using themethods or systems described herein, and the concentration of proteinswith molecular weight less than about 100 kDa can be less than about11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%,600%, 700%, 800%, 900%, or 1000% less than their concentration in abiological sample when not retained by the one or more filters using themethods or systems described herein. For example, the concentration ofA2M and other proteins with a molecular weight higher than 100 kDa canbe present at a concentration of at least about 1.5 times higher thantheir concentration in a biological sample after retention by one ormore filters using the methods or systems described herein, and theconcentration of proteins with molecular weight less than about 100 kDacan be less than about 10% less than their concentration in a biologicalsample when not retained by the one or more filters.

In some embodiments, an autologous composition can comprise an elevatedconcentration of A2M compared to the concentration of A2M found in abiological sample and an elevated concentration of one or more proteinswith molecular weight higher than 500 kDa found in a biological sample.The concentration of A2M in an autologous composition can be at leastabout 1.5 times higher than the concentration of A2M found in abiological sample and the concentration of one or more proteins withmolecular weight higher than 500 kDa can be at least about 1.5 timeshigher than the concentration of the one or more proteins with molecularweight higher than 500 kDa found in a biological sample. For example,the concentration of the concentration of A2M can be at least about 1.6,1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 500 kDa can be at least about1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 times higher than the concentration ofthe one or more proteins with molecular weight higher than about 500 kDafound in a biological sample. For example, the concentration of A2M canbe at least about 2 times higher than the concentration of A2M found ina biological sample and the concentration of one or more proteins withmolecular weight higher than 500 kDa can be at least about 2 timeshigher than the concentration of the one or more proteins with molecularweight higher than about 500 kDa found in a biological sample. Proteinswith a molecular weight higher than about 500 kDa can be, for example,proteins with a molecular weight higher than about 550 kDa, 600 kDa, 650kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa,1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa,1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa,1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, or higher.

The concentration of A2M and other proteins with a molecular weighthigher than 500 kDa can be present at a concentration of at least about1.5 times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein. For example, the concentration of A2M and other proteins with amolecular weight higher than 500 kDa can be present at a concentrationof at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein. For example, theconcentration of A2M and other proteins with a molecular weight higherthan 500 kDa can be present at a concentration of at least about 1.5times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein.

In some embodiments, the concentration of proteins with molecular weightless than about 500 kDa can be less than about 10% of the concentrationsof those proteins in a biological sample when not retained by the one ormore filters. For example, the concentration of proteins with molecularweight less than about 500 kDa can be less than about 11%, 12%, 13%,14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%,800%, 900%, or 1000% less than their concentration in a biologicalsample when not retained by the one or more filters. For example, theconcentration of proteins with molecular weight less than about 500 kDacan be less than about 20% less than their concentration in a biologicalsample when not retained by the one or more filters. Proteins with amolecular weight less than about 500 kDa can be, for example, proteinswith a molecular weight less than about 450 kDa, 400 kDa, 350 kDa, 300kDa, 250 kDa, 200 kDa, 150 kDa, 100 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa,30 kDa, 25 kDa, 20 kDa, 15 kDa, 10 kDa, 5 kDa, or less.

The concentration of A2M and other proteins with a molecular weighthigher than 500 kDa can be present at a concentration of at least about1.5 times higher than their concentration in a biological sample afterretention by one or more filters using the methods or systems describedherein and the concentration of proteins with molecular weight less thanabout 500 kDa can be less than about 10% of the concentrations of thoseproteins in a biological sample when not retained by the one or morefilters. For example, the concentration of A2M and other proteins with amolecular weight higher than 500 kDa can be present at a concentrationof at least about 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein, and theconcentration of proteins with molecular weight less than about 500 kDacan be less than about 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% less than theirconcentration in a biological sample when not retained by the one ormore filters. For example, the concentration of A2M and other proteinswith a molecular weight higher than 500 kDa can be present at aconcentration of at least about 1.5 times higher than theirconcentration in a biological sample after retention by one or morefilters using the methods or systems described herein, and theconcentration of proteins with molecular weight less than about 500 kDacan be less than about 10% less than their concentration in a biologicalsample when not retained by the one or more filters.

The concentration of A2M found in a biological sample, such as a bloodsample from a normal subject, can be between about 0.1 mg/mL to about 6mg/mL. For example, the concentration of A2M found in a blood samplefrom a normal subject or a normal biological sample can be between about0.1 mg/mL to 5.5 mg/mL, 0.1 mg/mL to 5 mg/mL, 0.1 mg/mL to 4.5 mg/mL,0.1 mg/mL to 4 mg/mL, 0.1 mg/mL to 3.5 mg/mL, 0.1 mg/mL to 3 mg/mL, 0.1mg/mL to 2.5 mg/mL, 0.1 mg/mL to 2 mg/mL, 0.1 mg/mL to 1.5 mg/mL, 0.1mg/mL to 1 mg/mL, 0.1 mg/mL to 0.75 mg/mL, 0.1 mg/mL to 0.5 mg/mL, 0.1mg/mL to 0.25 mg/mL, 1 mg/mL to 6 mg/mL, 1 mg/mL to 5.5 mg/mL, 1 mg/mLto 5 mg/mL, 1 mg/mL to 4.5 mg/mL, 1 mg/mL to 4 mg/mL, 1 mg/mL to 3.5mg/mL, 1 mg/mL to 3 mg/mL, 1 mg/mL to 2.5 mg/mL, 1 mg/mL to 2 mg/mL, 1mg/mL to 1.5 mg/mL, 2 mg/mL to 6 mg/mL, 2 mg/mL to 5.5 mg/mL, 2 mg/mL to5 mg/mL, 2 mg/mL to 4.5 mg/mL, 2 mg/mL to 4 mg/mL, 2 mg/mL to 3.5 mg/mL,2 mg/mL to 3 mg/mL, 2 mg/mL to 2.5 mg/mL, 3 mg/mL to 6 mg/mL, 3 mg/mL to5.5 mg/mL, 3 mg/mL to 5 mg/mL, 3 mg/mL to 4.5 mg/mL, 3 mg/mL to 4 mg/mL,3 mg/mL to 3.5 mg/mL, 4 mg/mL to 6 mg/mL, 4 mg/mL to 5.5 mg/mL, 4 mg/mLto 5 mg/mL, 4 mg/mL to 4.5 mg/mL, 5 mg/mL to 6 mg/mL, or 5 mg/mL to 5.5mg/mL.

In some embodiments, an autologous composition with an elevatedconcentration of A2M can be characterized by a reduction in theconcentration of or a change in the ratios of cytokines, chemokines,other immunomodulatory mediators, for example, cytokines, chemokines,other immunomodulatory mediators with a molecular weight less than about100 kDa. In some embodiments, an autologous composition with an elevatedconcentration of A2M can be characterized by a reduction in theconcentration of or a change in the ratios of cytokines, chemokines,other immunomodulatory mediators, for example, cytokines, chemokines,other immunomodulatory mediators with a molecular weight less than about500 kDa. Other immunomodulatory mediators can include peptides,proteins, DNA, RNA, carbohydrates, other small molecules, proteases, andother degradative proteins.

Cytokines, chemokines and other molecules can be involved ininflammation. Cytokines can be small cell-signaling protein moleculesthat are secreted by one or more cells and are a category of signalingmolecules that can be used in intercellular communication. Cytokines canbe classified as proteins, peptides, or glycoproteins, chemokines,interleukins, tumor necrosis factors (TNFs), monocyte chemoattractantproteins (MCPs), IL-1-like cytokines, gamma chain cytokines, beta chaincytokines, IL-6-like cytokines, IL-10-like cytokines, interferons, tumornecrosis factors, TGF-beta, macrophage inflammatory proteins (MIPs),tumor growth factors (TGFs), and matrix metalloproteases (MMPs). Forexample, cytokines can be interleukins, such as IL-1-like, IL-1α(hematopoietin-1), IL-1β (catabolin), IL-1RA (IL-1 receptor antagonist),IL-18 (interferon-γ inducing factor), Common g chain (CD132), IL-2 (Tcell growth factor), IL-4 (BSF-1), IL-7, IL-9 (T cell growth factorP40), IL-13 (P600), IL-15, Common b chain (CD131), IL-3 (multipotentialCSF, MCGF), IL-5 (BCDF-1), GM-CSF (CSF-2), IL-6-like, IL-6 (IFN-β2,BSF-2), IL-11 (AGIF), G-CSF (CSF-3), IL-12 (NK cell stimulatory factor),LIF (leukemia inhibitory factor), OSM (oncostatin M), IL-10-like, IL-10(CSIF), IL-20, IL-14 (HMW-BCGF), IL-16 (LCF), and IL-17 (CTLA-8);interferons, such as IFN-α, IFN-β, and IFN-γ, tumor necrosis factors(TNFs), such as CD154 (CD40L, TRAP), LT-β, TNF-α (cachectin), TNF-β(LT-α), 4-1BBL, APRIL (TALL-2), CD70 (CD27L), CD153 (CD3OL), CD178(FasL), GITRL, LIGHT, OX40L, TALL-1, TRAIL (Apo2L), TWEAK (Apo3L), andTRANCE (OPGL); tumor growth factors, such as TGF-β1, (TGF-β), TGF-β2,and TGF-β3; and hematopoietins, such as Epo (erythropoietin), Tpo(MGDF), Flt-3L, SCF (stem cell factor, c-kit ligand), M-CSF (CSF-1), andMSP (Macrophage stimulating factor). Other cytokines can include MST-1,CD40LG (TNFSF5), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL36RN,IL36A, IL37, IL36B, IL36G, IL22, IL5, IL8, IL9, LTA, LTB, MIF, AIMP1,SPP1, and TNF. Exemplary, cytokine receptors can be IFNA2, IL10RA,IL10RB, IL13, IL13RA1, IL5RA, IL9, and IL9R.

Chemokines can be a family of small cytokines, or proteins secreted bycells. Some chemokines can be pro-inflammatory and can be induced duringan immune response to recruit cells of an immune system to a site ofinfection, while others can be homeostatic and can be involved incontrolling the migration of cells during normal processes of tissuemaintenance or development. For example, chemokines can be XCL1(lymphoactin a, SCM-1a, ATAC), XCL2 (lymphoactin b, SCM-1b, ATAC) CCL1(1-309), CCL2 (MCP-1, MCAF), CCL3 (MIP-1α, LD78α), CCL4 (MIP-1β, LAG-1,ACT-2), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL11 (eotaxin),CCL13 (MCP-4), CCL14 (HCC-1), CCL15 (HCC-2, Lkn-1, MIP-1d, MIP-5), CCL16(HCC-4, LEC, LMC, LCC-1), CCL17 (TARC), CCL18 (DC-CK1, PARC, AMAC-a,MIP-4), CCL19 (MIP-3β, ELC, exodus-3), CCL20 (MIP-3α, LARC, exodus-1),CCL21 (6Ckine, SLC, exodus-2), CCL22 (MDC, STCP-1), CCL23 (MPIF-1,MIP-3, CKb-8), CCL24 (MPIF-2, eotaxin-2, CKb-6), CCL25 (TECK, MIP-4a),CCL26 (eotaxin-3), CCL27 (Eskine, CTACK, ILC), CXCL1 (GROa, MGSA-a),CXCL2 (GROb, MGSA-b, MIP-2a), CXCL3 (GROg, MGSA-g, MIP-2b), CXCL4 (PF4,oncostatin A), CXCL5 (ENA-78, CXCL6 (GCP-2), CXCL7 (NAP-2, PPBP), CXCL8(IL-8, NAP-1, NAF, MDNCF), CXCL9 (Mig), CXCL10 (IP-10), CXCL11(I-TAC),CXCL12 (SDF-1α/β), CXCL13 (BLC, BCA-1), CXCL14 (BRAK), CX3CL1(fractaline). Chemokine receptors can be CCL13 (mcp-4), CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, CXCR1, XCR1 (CCXCR1).Other proteins involved in inflammation can be ABCF1, BCL6, C3, C4A,CEBPB, CRP, CARD18, IL1R1, IL1RN, CXCR2, LTB4R, and TOLLIP.

Any of the autologous compositions described herein comprising A2M canfurther comprise one or more additional non-blood derived components.Non-blood derived components can be added before, during, or afterisolation by any of the methods described herein. A non-blood derivedcomponent can be obtained from non-blood tissues. A non-blood derivedcomponent can be an anti-coagulant. For example, a non-blood derivedcomponent or an anti-coagulant can be EDTA, tri-sodium citrate, waterfor injection (WFI), acid-citrate-dextrose (ACD),citrate-phosphate-dextrose (CPD), citrate-phosphate-double dextrose(CP2D), citrate-phosphate-dextrose-adenine (CPDA1), or saline.

Any of the autologous compositions described herein can comprise one ormore additional blood products or blood-derived components. Bloodproducts or blood-derived components can be added before, during, orafter isolation by any of the methods described herein. Blood productsor blood-derived components can be cells, peptides, proteins, DNA, RNA,carbohydrates, or other small molecules. For example, blood products orblood-derived components can be red blood cells, white blood cells,platelets, packed red blood cells, platelet-rich plasma, plateletconcentrates, fresh plasma, fresh frozen plasma, frozen plasma,cryoprecipitate and cryosupernant.

In some embodiments, an autologous composition can contain platelet richplasma (“PRP”). PRP is an autologous blood product that can be used inconjunction with autograft or allograft bone. The scientific rationalefor this clinical use is that PRP is a recognized material frequentlyused by orthopedic healthcare providers due to the ability of plateletconcentrates to release growth factors to the surgical site along withbone graft. Platelets can be prepared by any means known in the art. Thecellular components of PRP products generally include platelets withconcentrations that vary between 2 and 10-fold over whole blood. PRPproducts can also comprise variable concentrations of other growthfactors released upon platelet degranulation, including, but not limitedto, transforming growth factor-beta (TGF-β), insulin-like growthfactor-1 (IGF-1), vascular endothelial growth factor (VEGF), epidermalgrowth factor (EGF), fibroblast growth factor (FGF), and other factors.The growth factor content in various PRP products can vary between bothpatients and the method of preparation.

In some embodiments, an autologous composition can contain platelets.Typically, platelets can be prepared by separating platelets in bloodfrom other blood components. In some embodiments, platelets can beobtained from any of the methods or systems described herein. In someembodiments, PRP can be separated from whole blood via centrifugation.Centrifugation can be used to separate plasma and platelets, which canbe retained, from red and white blood cells, which can be discarded. Insome embodiments, centrifugation parameters can be designed to achieveplasma containing approximately 70-100% of the platelets contained inthe original blood sample, and to avoid the collection of leukocytes,such as less than 5%, for further processing. In some embodiments,concentrated platelets in blood plasma can be obtained by apheresis orpheresis (centrifugal separation during the donor process while othercomponents are returned to the donor) or by selective removal from wholeblood after gravity or centrifugal sedimentation of blood cells.

Though PRP preparations contain growth factors, other molecularmediators are also present, including cytokines, proteases and plasmaproteins. Some of these mediators are potentially pro-inflammatory orcatabolic, and are thought to be derived from leukocytes. Though all PRPpreparations contain concentrated platelets, leukocytes and other bloodderived cells may also be present and contribute to the molecularprofile of the PRP products. It is an object of the current invention toconcentrate platelets and, in some embodiments, allow for the retentionof platelet-released growth factors, while, in some embodiments, using amolecular weight cutoff of a filter and a tangential flowultrafiltration (TFF) step to avoid the concentration of potentiallyproinflammatory cytokines and catabolic proteases. After obtaining PRP,tangential flow ultrafiltration (TFF) can be used to concentrateplatelets to a desired concentration range using the methods describedherein, resulting in an autologous platelet integrated concentrate inwhich low molecular weight proteins, such as cytokines and proteases,such as those less than about 500 kDa in mass, have not beenconcentrated. In some embodiments, filter parameters can be chosen toconcentrate platelets but to avoid concentration of cytokines,proteases, and potentially undesirable plasma proteins.

Methods of A2M Enrichment and Preparation of Autologous Compositions

Methods of enrichment of A2M from a subject are also provided herein.The methods can be used to produce any of the autologous compositionsdescribed herein.

A method for enrichment of A2M from a biological sample, such as amammalian biological sample, can comprise flowing or passing abiological sample through one or more filters. Flowing or passing asample through one or more filters can comprise flowing the samplethrough 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more filters. A sample can beseparated into one or more filtrates and one or more retentates, forexample a first filtrate and a first retentate. For example, a samplecan be separated into 2, 3, 4, 5, 6, 7, 8, 9, 10, or more filtrates and2, 3, 4, 5, 6, 7, 8, 9, 10, or more retentates upon flowing or passingthe sample through one or more filters. For example, a sample can beseparated into 2 or more filtrates and 2 or more retentates upon flowingor passing the sample through two or more filters. An A2M enrichedsample can be a first, second, third, fourth, fifth, or more retentateupon flowing or passing a biological sample through one or more filters.One or more A2M enriched samples or retentates can be diluted, such aswith a diluent. A diluent can be a liquid or a solution, such as ahypotonic, hypertonic, or isotonic solution. For example, a diluent canbe a WFI solution or a saline solution.

A method for enrichment of A2M from a biological sample, such as amammalian biological sample can comprise separating cells from acellular biological sample, such as blood. In some embodiments, redblood cells can be separated from white blood cells and platelets byperforming one or more centrifugation steps. White blood cells can beseparated from platelets by performing one or more centrifugation steps.A supernatant of a centrifuged blood sample can contain A2M. In someembodiments, the supernatant can contain A2M and platelets. In someembodiments, the supernatant can contain A2M and not contain platelets.

In some embodiments, it is preferable to separate red blood cells andwhite blood cells from the biological sample and platelets within thebiological sample prior to flowing the sample through one or morefilters by performing one or more centrifugation steps. In someembodiments, it is preferable to separate red blood cells, white bloodcells, and platelets from the biological sample prior to flowing orpassing the sample through the one or more filter by performing one ormore centrifugation steps.

In some embodiments, it is preferable to separate red blood cells andwhite blood cells from the biological sample and platelets within thebiological sample by flowing or passing the biological sample throughone or more filters. In some embodiments, it is preferable to separatered blood cells, white blood cells, and platelets from the biologicalsample by flowing or passing the biological sample through one or morefilters. The one or more filters used to remove the cells and otherlarge particles from the biological sample can have a pore size of atleast about 0.1 μm. For example, the one or more filters used to removethe cells and other large particles from the biological sample can havea pore size of at least about 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm,0.7 μm, 0.7 μm, 0.8 μm, 0.9 μm, lμm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, or 3 μm, or higher. As anon-limiting example, a biological sample, such as blood, can be flowedthrough one or more filters with a pore size of at least about 0.2 μmwherein the red blood cells and white blood cells are retained by thefilter and are in the retentate and non-cellular components andplatelets are not retained by the filter and are in the filtrate. Asanother non-limiting example, a biological sample, such as blood, can beflowed through one or more filters with a pore size of at least about0.2 μm wherein the red blood cells, white blood cells, and platelets areretained by the filter in the retentate and non-cellular components arenot retained by the filter and are in the filtrate.

In some embodiments, one or more of the filters can have a charge,immobilized molecules, or a combination thereof and can thereby enhancethe selectivity of the filters. Immobilized molecules can be antibodies,proteins, receptors, ligands, carbohydrates, nucleotides, RNA, DNA orany combination thereof. For example, enhancing the selectivity offilters can enhance the ability of a filter to retain A2M in theretentate upon flowing or passing a biological sample through the filteror, as another example, enhance the ability of a filter to not retainmolecules that are not A2M upon flowing a biological sample through thefilter.

Additionally, one or more filters with a molecular weight cut-off can beused and can allow a percentage of particles in the biological sample,such as cells and proteins, with a molecular weight higher than themolecular weight cut-off of the filter to be retained by the filter. Theretained sample can be a retentate and the sample that flows through thefilter can be a filtrate. A filter with a molecular weight cut-off canallow less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% ofparticles in a filtrate, such as cells and proteins, with a molecularweight higher than the molecular weight cut-off of the filter to beretained by the filter. For example, one or more filters can remove 100%of cells, cellular debris, or a combination thereof, from a blood sampleand can remove or reduce the ratio of proteins with a molecular weightless than 500 kDa from a biological sample containing A2M relative toA2M concentration.

One or more filtrates obtained from the methods described herein can bepassed through one or more other filters by applying a gravitational,centrifugal, or mechanical force to the one or more filtrates. A samplecan be separated into one or more other filtrates and one or more otherretentates, for example a second filtrate and a second retentate. Forexample, a sample can be separated into 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore other filtrates and 2, 3, 4, 5, 6, 7, 8, 9, 10, or more otherretentates upon flowing the sample through one or more other filters.For example, a sample can be separated into 2 other filtrates and 2other retentates upon flowing or passing the sample through one or moreother filters. An A2M enriched sample can be a first, second, third,fourth, fifth, or more other retentate upon flowing a biological samplethrough one or more other filters. One or more other retentates,filtrates, or A2M enriched samples can be diluted, such as with adiluent. For example, a diluent can be a liquid, such as a WFI solutionor a saline solution.

In some embodiments, after separating the cells from a biological sampleaccording to the methods described herein, the resulting biologicalsample, lacking red blood cells, white blood cells and platelets, can beflowed or passed through one or more filters to obtain one or morefiltrates and retentates, such as an A2M enriched retentate or A2Mconcentrated retentate. The resulting biological sample lacking redblood cells, white blood cells and platelets can be flowed through oneor more filters with a molecular weight cut-off of at most about 500 kDato obtain one or more filtrates and retentates, such as an A2M enrichedor concentrated retentate. For example, the resulting biological samplelacking red blood cells, white blood cells and platelets can be flowedthrough one or more filters with a molecular weight cut-off of at mostabout 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490kDa or lower to obtain one or more filtrates and retentates, such as anA2M enriched retentate or A2M concentrated retentate.

A retentate, such as an A2M enriched or concentrated retentate obtainedby flowing or passing a biological sample lacking red blood cells, whiteblood cells and platelets through one or more filters with a molecularweight cut-off of at most about 500 kDa, can comprise an elevatedconcentration of A2M compared to the concentration of A2M found in abiological sample and an elevated concentration of one or more proteinswith molecular weight higher than 500 kDa found in a biological sample.The concentration of A2M in a retentate obtained by flowing a biologicalsample lacking red blood cells, white blood cells and platelets throughone or more filters with a molecular weight cut-off of at most about 500kDa can be at least about 1.5 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 500 kDa can be at least about1.5 times higher than the concentration of the one or more proteins withmolecular weight higher than 500 kDa found in a biological sample. Forexample, the concentration of A2M in a retentate obtained by flowing abiological sample lacking red blood cells, white blood cells andplatelets through one or more filters with a molecular weight cut-off ofat most about 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480kDa, 490 kDa, 500 kDa, or lower, can be at least about 1.6, 1.7, 1.8,1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10,10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000 times higher than the concentration of A2M found in abiological sample and the concentration of one or more proteins withmolecular weight higher than 500 kDa can be at least about 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of theone or more proteins with molecular weight higher than about 500 kDafound in a biological sample. For example, the concentration of A2M in aretentate obtained by flowing a biological sample lacking red bloodcells, white blood cells and platelets through one or more filters witha molecular weight cut-off of at most about 500 kDa can be at leastabout 2 times higher than the concentration of A2M found in a biologicalsample and the concentration of one or more proteins with molecularweight higher than 500 kDa can be at least about 2 times higher than theconcentration of the one or more proteins with molecular weight higherthan about 500 kDa found in a biological sample.

In some embodiments, after separating the cells from a biological sampleaccording to the methods described above, the resulting biologicalsample lacking red blood cells and white blood cells, but not lackingplatelets, can be flowed through one or more filters to obtain one ormore filtrates and retentates, such as an A2M enriched or concentratedretentate. The resulting biological sample lacking red blood cells,white blood cells, but not lacking platelets, can be flowed through oneor more filters with a molecular weight cut-off of at most about 500 kDato obtain one or more filtrates and retentates, such as an A2M enrichedor concentrated retentate. For example, the resulting biological samplelacking red blood cells and white blood cells, but not lackingplatelets, can be flowed through one or more filters with a molecularweight cut-off of at most about 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460kDa, 470 kDa, 480 kDa, 490 kDa or lower to obtain one or more filtratesand retentates, such as an A2M enriched or concentrated retentate.

A retentate, such as an A2M enriched or concentrated retentate obtainedby flowing or passing a biological sample lacking red blood cells andwhite blood cells, but not lacking platelets, through one or morefilters with a molecular weight cut-off of at most about 500 kDa, cancomprise an elevated concentration of A2M compared to the concentrationof A2M found in a biological sample and an elevated concentration of oneor more proteins with molecular weight higher than 500 kDa found in abiological sample. The concentration of A2M in a retentate obtained byflowing a biological sample lacking red blood cells and white bloodcells, but not lacking platelets, through one or more filters with amolecular weight cut-off of at most about 500 kDa can be at least about1.5 times higher than the concentration of A2M found in a biologicalsample and the concentration of one or more proteins with molecularweight higher than 500 kDa can be at least about 1.5 times higher thanthe concentration of the one or more proteins with molecular weighthigher than 500 kDa found in a biological sample. For example, theconcentration of A2M in a retentate obtained by flowing a biologicalsample lacking red blood cells and white blood cells, but not lackingplatelets, through one or more filters with a molecular weight cut-offof at most about 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa,160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa,240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa,320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa,400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa,480 kDa, 490 kDa, 500 kDa, or lower, can be at least about 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times higher than the concentration of A2Mfound in a biological sample and the concentration of one or moreproteins with molecular weight higher than 500 kDa can be at least about1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400,500, 600, 700, 800, 900, or 1000 times higher than the concentration ofthe one or more proteins with molecular weight higher than about 500 kDafound in a biological sample. For example, the concentration of A2M in aretentate obtained by flowing a biological sample lacking red bloodcells, white blood cells, but not lacking platelets, through one or morefilters with a molecular weight cut-off of at most about 500 kDa can beat least about 2 times higher than the concentration of A2M found in abiological sample and the concentration of one or more proteins withmolecular weight higher than 500 kDa can be at least about 2 timeshigher than the concentration of the one or more proteins with molecularweight higher than about 500 kDa found in a biological sample.

In some embodiments, after passing a sample through one or more filtersaccording to the methods described herein, at least about 10% ofparticles, and proteins with a molecular weight less than 500 kDa can beremoved from the sample. For example, at least about 11%, 12%, 13%, 14%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% of cells, particles, and small proteins with amolecular weight less than 500 kDa can be removed from the sample. Forexample, at least about 20% of particles and proteins with a molecularweight less than 500 kDa can be removed from the sample. At least about11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells, particles, andproteins with a molecular weight less than about 100 kDa, 110 kDa, 120kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, or less can be removedfrom the sample. For example, at least about 20% of particles and smallproteins with a molecular weight less than 500 kDa, can be removed fromthe sample. An autologous composition described herein can be isolatedafter passing a sample through one or more filters according to themethods described herein.

In some embodiments, after passing a biological sample through one ormore filters according to the methods described herein, at least about10% of cells, particles, and small proteins with a molecular weight lessthan 100 kDa can be removed from the sample For example, at least about11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells, particles, andsmall proteins with a molecular weight less than 100 kDa can be removedfrom the sample. For example, at least about 20% of cells, particles,and small proteins with a molecular weight less than 100 kDa can beremoved from the sample. At least about 11%, 12%, 13%, 14%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% of cells, particles, and small proteins with a molecularweight less than 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa,60 kDa, 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa,15 kDa, 10 kDa, 5 kDa, or less can be removed from the sample. Forexample, at least about 20% of cells, particles, and small proteins witha molecular weight less than 100 kDa, can be removed from the sample. Anautologous composition described herein can be isolated after passing asample through one or more filters according to the methods describedherein.

One or more centrifugation cycles can be used or applied to providecentrifugal force to flow or push a biological sample through one ormore filters. Gravitational, centrifugal or mechanical force can also beused or applied to provide force to flow or push a biological samplethrough one or more filters. Mechanical force can be a pump, centrifugalforce, gas pressure, or any other force that is operable to provideenough force to flow a sample through one or more filters as describedherein.

Filters can be positively charged, negatively charged, or not charged.Filters can be made of Polyesteramide (Nylon), Mixed Cellulose Ester(MEC), Polyfluortetraethylene, Polyether sulfone, PolyvinylideneFluoride (PVDF), Phosphocellulose (PH), DEAE (DE), Polypropylene,Cellulose Acetate, Glass Fiber, or any combination thereof.

Filters can be characterized by a molecular weight cut-off. For example,a filter can be characterized as having a molecular weight cut-off of500 kDa or less, such as about 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460kDa, 470 kDa, 480 kDa, 490 kDa, or less.

A filter with a molecular weight cut-off can allow a large percentage ofparticles in a sample, such as cells and proteins, with a molecularweight less than the molecular weight cut-off of the filter to passthrough the filter. The sample that flows through the filter can be thefiltrate. For example, a filter with a molecular weight cut-off canallow more than about 5%, 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of particles in asample, such as cells and proteins, with a molecular weight less thanthe molecular weight cut-off of the filter to pass through the filter.For example, a filter with a molecular weight cut-off can allow morethan about 50% of particles in a sample, such as cells and proteins,with a molecular weight less than the molecular weight cut-off of thefilter to pass through the filter.

After passing one or more filtrates through one or more other filtersaccording to the methods described herein, at least about 10% of cells,particles, and small proteins with a molecular weight less than 500 kDacan be removed from a filtrate. For example, at least about 11%, 12%,13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of cells, particles, and small proteinswith a molecular weight less than 500 kDa can be removed from afiltrate. For example, at least about 20% of cells, particles, and smallproteins with a molecular weight less than 500 kDa can be removed from afiltrate. For example, at least about 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% of cells, particles, and small proteins with a molecular weightless than about 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa,400 kDa, or 450 kDa can be removed from a filtrate. For example, atleast about 20% of cells, particles, and small proteins with a molecularweight less than about 500 kDa can be removed from a filtrate. Afterpassing a filtrate through one or more filters according to the methodsdescribed herein, a composition described herein can be isolated.

One or more additional non-blood derived components can be added to theone or more filtrates or retentates. Non-blood derived components can beadded before, during, or after isolation by any of the methods describedherein. A non-blood derived component can be an anti-coagulant. Forexample, an anti-coagulant can be EDTA, tri-sodium citrate, water forinjection (WFI), or saline.

One or more additional blood products or blood-derived components can beadded to the one or more filtrates or retentates. Blood products orblood-derived components can be added before, during, or after isolationby any of the methods described herein. Blood products or blood derivedcomponents can be cells, peptides, proteins, DNA, RNA, carbohydrates, orother small molecules. For example, blood products or blood-derivedcomponents can be red blood cells, white blood cells, or platelets.

Platelets can be isolated from a biological sample according to anymethod known in the art, such as by centrifugation of a blood sample.Red blood cells and white blood cells can be sedimented bycentrifugation at relatively low centrifugal force, for example, lessthan 1000 g. Platelets can be isolated by centrifugation of the plateletcontaining plasma obtained from a first centrifugation. A plateletcontaining plasma can be centrifuged, for example, between about 3000 gto 5000 g, to sediment platelets. The above procedure can also beperformed in one centrifugation step.

In some embodiments, one or more other filtrates or retentates, forexample a second filtrate or second retentate can be collected. One ormore additional non-blood derived components can be added to the one ormore other filtrates or other retentates. Non-blood derived componentscan be added before, during, or after isolation by any of the methodsdescribed herein. A non-blood derived component can be ananti-coagulant. For example, an anti-coagulant can be EDTA, tri-sodiumcitrate, water for injection (WFI), or saline.

One or more additional blood products or blood-derived components can beadded to the one or more other filtrates or other retentates. Bloodproducts or blood-derived components can be added before, during, orafter flowing or passing the biological sample through one or morefilters according to any of the methods described herein. Blood productsor blood-derived components can be cells, peptides, proteins, DNA, RNA,carbohydrates, or other small molecules. For example, blood products orblood-derived components can be red blood cells, white blood cells, orplatelets.

Systems for Production of Autologous A2M Compositions

Also provided herein are systems that can be used with the methodsdescribed herein and can be used to produce any of the compositionsdescribed herein. A system for enrichment of A2M from a mammalian sampleis provided. A system can have one or more filters, a centrifuge, apump, or a combination thereof. A system can have one or more waste orpermeate collection modules.

One aspect of the invention is directed at a system for concentratingA2M from a fluid sample. Typically, the fluid sample is blood derivedfrom a patient and the system concentrates the A2M from the blood into aconcentrated A2M blood serum or concentrated A2M blood plasma. Anexemplary embodiment of the system comprises a filtration modulecomprising an inlet and an outlet and one or more filters. A flow of thefluid sample flows into the filtration module and through at least theinlet and one or more filters of the filtration module. The flow mayalso pass through the outlet of the module after passing through theinlet and one or more filters. The one or more filters are typicallyconnected in series between the inlet and the outlet of the filtermodule. The inlet and outlet may have selectively closed valves tocontrol flow of the fluid sample therein and the module may comprisemultiple inlets or multiple outlets or any combination thereof. Thefiltration module may be a dead-end filtration module. Alternatively,the filtration module may be a tangential flow filtration module.

In some embodiments the one or more filters of the filtration modulecomprise at least a first and a second filter. The first filter screensout cells, particles, and other molecules larger than 1 micron. Thesecond filter screens out molecules having a weight less than about 500kDa. The second filter may also retain molecules having a weight of morethan about 500 kDa. In some embodiments the first and the second filterscomprise cross-flow filters.

Some embodiments of the invention further comprise a pump adapted to befluidly coupled with the filtration module either upstream of the inletof the filtration module or downstream of the outlet of the filtrationmodule. The pump is further adapted to produce a flow of the fluidsample that passes through the one or more filters of the filter module.In some embodiments of the invention, the filter module furthercomprises at least one reservoir.

In an exemplary embodiment of the invention wherein the first filtercomprises a cross-flow filter that screens out cells particles and othermolecules lager than 1 micron. A retentate of this filter containing thecells, particles, and other molecules larger than 1 micron of the fluidsample is stored in a first retentate reservoir. Alternatively, theretentate of the first filter is discarded. A permeate of the firstfilter is directed to a first permeate reservoir, the first permeatereservoir is then typically connected to the second filter. The permeateof the first filter flows through the second filter. The second filtermay typically be a cross flow filter adapted to retain molecules ofweight more than about 500 kDa. A retentate of the second filtercomprises these molecules of weight more than about 500 kDa may beretained in the first permeate collection reservoir. The retentate ofthe second filter typically comprises the concentrated A2M of the fluidsample. A permeate of the second filter may directed to a separatesecond filter permeate reservoir. Alternatively, the permeate of thesecond reservoir may be redirected through the outlet of the filtrationmodule and circulated back to the inlet of the filtration module suchthat the fluid sample is processed by the filtration module multipletimes or continuously.

In other exemplary embodiments of the invention the system forconcentrating A2M further comprises a centrifuge. The fluid sample iscentrifuged to produce a supernatant and a pellet, the supernatantcontaining small molecules and A2M but not large particles such ascells. The pellet contains the large particles such as cells present inthe fluid sample. The supernatant is then directed through thefiltration module. The filtration module comprising at least one filteradapted to retain molecules of weight more than about 500 kDa. The atleast one filter typically comprises a 500 kDa cross flow filter asdescribe above. The retentate of the 500 kDa cross flow filter typicallycomprising the A2M of the supernatant is retained in a retentatereservoir. The permeate of the at least one filter may be directed to awaste reservoir or discarded. Alternatively, the permeate of the atleast one filter may be directed to the filter module outlet where it isredirected to the filter module inlet such that the supernatant of thefluid sample passes through the filter module multiple times.

Another aspect of the invention comprises a method of concentrating A2Mfrom a fluid sample comprising providing a filtration module. Thefiltration module comprises an inlet, an outlet and one or more filtersfluidly connected in series between the inlet and the outlet. The methodfurther comprises removing cells from the fluid sample. The method alsofurther comprises pumping the fluid sample through the filtration moduleinlet and the one or more filters to produce a concentrated A2M serum orplasma. The fluid sample may also be pumped through the outlet. Pumpingthe fluid sample is accomplished with a pump fluidly connected to thefiltration module either upstream of the inlet or downstream of theoutlet. The one or more filters of the filtration module comprises atleast one 500 kDA filter configured to retain molecules of weight morethan about 500 kDa.

In some embodiments of the method described above, removing cells fromthe fluid sample comprises providing a centrifuge, centrifuging thefluid sample, and obtaining a resultant supernatant of the fluid sample.A resultant pellet of the fluid sample comprising cells and largemolecules may be retained. The resultant supernatant of the fluid sampletypically comprises A2M and small molecules but not cells and largemolecules. The supernatant of the fluid sample is then pumped throughthe filtration module to concentrate the A2M. The 500 kDa filter of thefiltration module is typically a 500 kDa cross-flow filter, a retentateof the 500 kDa cross-flow filter is retained and comprises theconcentrated A2M serum.

In some embodiments, removing cells from the fluid sample comprisesfiltering the fluid sample with a first filter of the filtration moduleadapted to screen out cells, particles, and molecules larger than 1micron. Typically the first filter is a cross-flow filter and a permeateof the first filter is directed to the at least one 500 kDa filter ofthe filtration module. The permeate of the first filter may be stored ina first permeate reservoir. The 500 kDa filter of the filtration moduleis typically a 500 kDa cross-flow filter, a retentate of the 500 kDacross-flow filter is retained in the first permeate reservoir andcomprises the concentrated A2M serum. A permeate of the 500 kDa crossflow filter may be stored, discarded, or directed to the inlet of thefiltration module such that it be further filtered.

It should be understood that features of the above described method andsystem embodiments may be combined and interchanged with one another.

Cells and particles with a size of 0.6 μm or more, and other moleculeswith a molecular weight of 500 kDa or less can be removed from thesample by flowing or passing a sample through one or more filterscontained within the system in sequence. Removed cells and particles canbe disposed of or collected in a waste module. For example, cells andparticles of 0.6 μm, 1 μm, 2 μm, or 3 μm or more, and other moleculeswith a molecular weight of 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa,150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa,230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa,310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa,390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa,470 kDa, 480 kDa, 490 kDa, or less can be removed by the one or morefilters and can be deposited into one or more waste modules. A samplecan be flowed through one or more filters by applying centrifugal forceusing the centrifuge of the system, using the pump of the system, or acombination thereof. A system can further comprise a collection module.A retentate or filtrate can be collected or isolated in the collectionmodule, for example a retentate with an A2M enriched sample can beisolated in a collection module, after passing a sample through one ormore filters. A system can further comprise a sample loading module. Asample loading module can be operable to introduce the sample into thesystem. A sample loading module can be directly or indirectly attachedto the blood stream of a subject.

The first step in the system can be either a centrifugation step orfiltration step. The collected blood can be centrifuged at a particularcentrifugal force that allows the precipitation of the red blood cellsand white blood cells and other particles and debris, allowing plasmaproteins and platelets in the supernatant. This process can also beachieved by filtration using a hollow fiber membrane that will allow theplasma proteins and platelets to go through the membrane into thefiltrate and prevent the red blood cells and white blood cells and otherparticles and debris to remain in the retentate. In some embodiments,the supernatant from the centrifugation step or the filtrate from thefiltration step can be filtered on the second filter where proteins 500kDa or larger can be retained by the filter and smaller proteins than500 kDa and other molecules can pass through the membrane into thefiltrate. In some embodiments, the supernatant from the centrifugationstep or the filtrate from the filtration step can be filtered on thesecond filter where proteins 100 kDa or larger can be retained by thefilter and smaller proteins than 100 kDa and other molecules can passthrough the membrane into the filtrate. The concentrated retentate canthen be collected and injected into the patient.

In some embodiments, a collection receptacle with platelets and plasmacan be connected to any of the systems described herein, such as withhematologic tubing, and passed through a filter, such as hollow fibertangential flow filter (HFTFF), that uses a molecular weight cutoffmembrane, such as a 500 kDa molecular weight cutoff membrane, ofmodified polyethersulfone using a pump, such as a peristaltic pump. Insome embodiments, the flow-through port of the filter can be connectedusing hematologic tubing back to the collection bottle in a closed-loop.In some embodiments, the filtrate port of the filter can also beconnected using tubing connected to waste. In some embodiments, nopriming of the flow-circuit is necessary.

As a non-limiting example, a system, such as an APIC system (AutologousPlatelet Integrated Concentrate system) can be run until the plasmareaches between 2-10 times the concentrations as found in whole blood,for a total remaining volume of approximately 4-6 mL. In someembodiments, this process or a similar process can be performed inapproximately 10 to 30 minutes, 15 to 35 minutes, 20 to 40 minutes, or30 to 45 minutes. The total waste volume of approximately 36 mL,containing low molecular weight proteins, including potentiallypro-inflammatory cytokines and proteases, can be discarded. Theresulting autologous platelet concentrate can be drawn into a syringeand provided for mixing as needed for clinical administration withautograft or allograft bone. TFF is a filtration process whereby thesolution is constantly flowing over the membrane to prevent pores frombecoming clogged by cells and proteins. As used in the systems andmethods described herein, TFF discourages platelets and other largeproteins from blocking the membrane pores and allowing the flow of smallmolecules and proteins. The use of hollow fiber membranes can increasethe surface area that is available for filtration. The 500 kDa molecularweight cutoff of the membrane permits small molecules and proteins suchas cytokines, chemokines, and proteases to pass through, eventuallyleading to waste, but retaining larger particles (>500 kDa) such asplatelets.

FIG. 24 shows an embodiment of the filtration module of the system forconcentrating A2M. This particular embodiment of the filtration moduleis well suited to receive a supernatant 2409 of a fluid sample such asblood (not shown) that has been centrifuged. The filter module 2401 hasa first filter 2410 coupled to the filter module inlet 2402. Thesupernatant is 2409 received and pumped from a receiving reservoir 2405into the first filter. The first filter 2410 is typically a cross-flowfilter configured to retain molecules larger than about 500 kDa in aretentate reservoir 2406. The retentate reservoir 2406 may also be thesame as the receiving reservoir 2405 for receiving the supernatant 2409of the fluid sample. The permeate 2420 of the first filter 2410containing molecules smaller than about 500 kDa is directed to apermeate reservoir 2430 which may be a waste bag 2431. A2M isconcentrated in this retentate reservoir 2406. A filter module outlet2450 maybe coupled to the retentate reservoir 2406 such that theconcentrated A2M 2460 may be extracted from the retentate reservoir 2406or pumped back through the first filter 2410.

FIG. 28 shows an embodiment of the system for concentrating A2M. A bloodbag 2801 is shown containing the fluid sample 2802, which typicallycomprises blood. The fluid sample 2802 is extracted via syringe(s) 2803and centrifuged with centrifuge 2804. The resultant supernatant 2805containing A2M and other small molecules but not cells or largemolecules is then directed to the filtration module 3209, where in theA2M is concentrated in to a serum 2810 or a plasma 2811 with at leastone filter 2808 selected to retain molecules of larger in size thanabout 500 kDa. The filtration module 3209 may be similar to the exampledescribed in FIG. 24.

FIG. 32 shows an embodiment of the system for concentrating A2M. A bloodbag 3201 is shown containing the fluid sample 3202. The fluid sample3202 is pumped via pump 3203 to the first filter 3210 of the filtrationmodule 3204. The first filter 3210 shown here is a cross-flow filterconfigured to screen out cells, particles, and other molecules largerthan 1 micron. The permeate 3212 of the first filter comprisingcomponents of the fluid sample smaller than 1 micron is directed to afirst permeate reservoir 3215. The retentate 3216 of the first filter isdirected to a first retentate reservoir 3216, in this particularembodiment the first retentate reservoir is also blood bag 3201. Thepermeate 3212 of the first filter is then directed via pump 3203 to thesecond filter 3220. The second filter is typically a cross-flow filterconfigured to retain molecules of weight more than about 500 kDa.Molecules of weight more than about 500 kDa are retained as a secondretentate 3223 in a second retentate reservoir 3225. The secondretentate reservoir 3225 may be the same as the first permeate reservoir3215. Permeate of the second filter 3224 is typically directed to asecond permeate reservoir 3226 in some embodiments the second permeatereservoir 3226 is a waste bag 3230. The retentate of the second filter3223 comprises the concentrated A2M. The pump 3203 may be fluidlyconnected to the filtration module up stream of the filtration moduleinlet 3206 or down-stream of the filtration module outlet 3207 orin-between inlet the filtration module 3206 and the filtration moduleoutlet 3207 or any combination thereof. The second retentate reservoir3225 has an access port 3229 for directing flow of the concentrated A2M(which is also the retentate of the second filter) back to the pump.Such port may be used to access the concentrated A2M, or the permeate ofthe first filter, or the retentate of the second filter for subsequentprocessing or harvesting of the concentrated A2M.

Variant A2M Polypeptides Compositions for Treatment of Pain andInflammation

A2M (FIG. 4) is a general inhibitor of metalloproteases and otherproteases such as ADAMTS 4 and ADAMTS 5. These proteases and othersproduced as a result of or prior of degeneration and inflammation can beresponsible for cartilage and disc degeneration and pain in synovialjoints, the spine, tendons and ligaments, and other joints, entheses andgeneral tissues. Any of the recombinant compositions described hereincan be used for treatment of a subject with a condition, disease, painor inflammation according to any of the methods described herein.

A2M is able to inactivate an enormous variety of proteases (includingserine-, cysteine-, and aspartic-metalloproteases). A2M can function asan inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. A2Mcan function as an inhibitor of coagulation by inhibiting thrombin.Human A2M has in its structure a 38 amino acid “bait” region. The baitregion varies widely in the amino acid number (27-52 amino acids) andsequence between animal species. Proteases binding and cleaving of thebait region can become bound to A2M. The protease-A2M complex can berecognized by macrophage receptors and cleared from the organism'ssystem. A2M is able to inhibit all four classes of proteases by a unique‘trapping’ mechanism. When a protease cleaves the bait region, aconformational change can be induced in the protein which can trap theprotease. The entrapped enzyme can remain active against low molecularweight substrates (activity against high molecular weight substrates canbe greatly reduced). Following cleavage in the bait region a thioesterbond can be hydrolyzed and can mediate the covalent binding of theprotein to the protease.

In one aspect, provided herein is a composition that can be a variantA2M polypeptide. A variant A2M polypeptide can be a recombinant protein,or fragments thereof, and can be produced in a host cell and purifiedfor use in treatment of pain and inflammation conditions and diseases. Avariant A2M composition can be more efficient in inhibiting proteases,have longer half-life, have a slower clearance factor, or anycombination thereof compared to a wild-type A2M. A variant A2M can be arecombinant protein, or a fragment thereof, and can be produced in ahost cell and purified. For example, a variant A2M recombinant proteincan be produced in a host comprising bacteria, yeast, fungi, insect, ormammalian cells, or a cell free system.

Variant A2M polypeptides or fragments thereof, can also be variants orposttranslationally modified variants of A2M. A2M variant polypeptidescan have an integer number of amino acid alterations such that theiramino acid sequence shares at least about 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99%, 99.5% or 100% identity with an amino acid sequence of awild type A2M polypeptide. In some embodiments, A2M variant polypeptidescan have an amino acid sequence sharing at least about 60%, 70%, 80%,85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity with the amino acidsequence of a wild type A2M polypeptide.

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The Smith Waterman algorithm can also be used to determine percentidentity. Exemplary parameters for amino acid sequence comparisoninclude the following: 1) algorithm from Needleman and Wunsch (J. Mol.Biol., 48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoffand Hentikoff (Proc. Nat. Acad. Sci. USA., 89:10915-10919 (1992)) 3) gappenalty=12; and 4) gap length penalty=4. A program useful with theseparameters can be publicly available as the “gap” program (GeneticsComputer Group, Madison, Wis.). The aforementioned parameters are thedefault parameters for polypeptide comparisons (with no penalty for endgaps). Alternatively, polypeptide sequence identity can be calculatedusing the following equation: % identity-(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps

Variant A2M polypeptides, or fragments thereof, include but are notlimited to, those containing as a primary amino acid sequence all orpart of the amino acid sequence encoded by SEQ ID NOs: 5-66, andfragments of these proteins, including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. The variant A2Mpolypeptides can include all or part of the amino acid sequence encodedby SEQ ID NO: 3. The variant A2M polypeptides can be, for example, anynumber of between 4-20, 20-50, 50-100, 100-300, 300-600, 600-1000,1000-1450 consecutive amino acids containing the amino acids sequencesof SEQ. ID NOs. 5-66. The variant A2M polypeptide can be less than orequal to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400,500, 600, 700, 800, 900, 1000, and 1450 amino acids in length andcontain, as part of the sequence: SEQ ID NOs: 5-66. Variant A2Mpolypeptides includes polypeptide sequences having at least 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%,79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,65%, 64%, 63%, 62%, 61%, or 60% sequence identity or similarity to anyvariant A2M polypeptide containing one of SEQ ID NOs: 5-66.

The variant A2M polypeptides provided herein also include proteinscharacterized by amino acid sequences similar to those of purifiedproteins but into which modification are naturally provided ordeliberately engineered. For example, modifications, in the variant A2Mpeptide or variant A2M DNA sequence, can be made by those skilled in theart using known techniques. Modifications of interest in the proteinsequences can include the alteration, substitution, replacement,insertion or deletion of a selected amino acid residue in the codingsequence. For example, one or more of the cysteine residues can bedeleted or replaced with another amino acid to alter the conformation ofthe molecule. Techniques for such alteration, substitution, replacement,insertion or deletion are well known to those skilled in the art (see,e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration,substitution, replacement, insertion or deletion retains the desiredactivity of the protein. Regions of the protein that are important forthe protein function can be determined by various methods known in theart including the alanine-scanning method which involves systematicsubstitution of single or multiple amino acids with alanine, followed bytesting the resulting alanine-containing variant for biologicalactivity. This type of analysis can be used to determine the importanceof the substituted amino acid(s) in biological activity.

The bait region of A2M is a segment that is susceptible to proteolyticcleavage, and which, upon cleavage, initiates a conformational change inthe A2M molecule resulting in the collapse of the structure around theprotease. For the exemplary A2M sequences set forth in SEQ ID NO: 3, thebait region corresponds to amino acids 690-728. For the exemplary A2Msequences set forth in SEQ ID NO: 1 and 2, the bait region correspondsto the nucleotides encoding amino acids 690-728.

A variant A2M polypeptide can comprise a bait region of a variant A2Mpolypeptide. For example, a bait region of a variant A2M polypeptide canbe a mutant bait region, fragment of a bait region, a bait region fromanother species, an isoform of a bait region, or a bait regioncontaining multiple copies of one or more bait regions described herein,or any combination thereof. A bait region of a variant A2M polypeptidecan include a plurality of protease recognition sites arranged in seriesand can be arranged in any order.

A bait region of a variant A2M polypeptide can have one or more proteaserecognition sites. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moreprotease recognition sites. Protease recognition sites or substrate baitregions can be consensus sequences for serine proteases, threonineproteases, cysteine proteases, aspartate proteases, metalloproteases,glutamic acid proteases, or any combination thereof compared to a wildtype A2M protein. A variant A2M polypeptide can be characterized by anenhanced specific inhibition of serine proteases, threonine proteases,cysteine proteases, aspartate proteases, metalloproteases, glutamic acidproteases, or any combination thereof. A variant A2M polypeptide can becharacterized by an enhanced nonspecific inhibition of serine proteases,threonine proteases, cysteine proteases, aspartate proteases,metalloproteases, glutamic acid proteases, or any combination thereofcompared to a wild type A2M protein.

A bait region of a variant A2M polypeptide can have one or more mutantbase regions. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant baseregions. A bait region of a variant A2M polypeptide can have one or morebait region fragments. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or morebait region fragments. A fragment of a bait region of a variant A2Mpolypeptide can be a fragment of any of SEQ ID NOs: 5-66.

A bait region of a variant A2M polypeptide can have one or more mutantamino acids that are different than those amino acids in a wild-type A2Mpolypeptide. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant amino acidsthat are different than those amino acids in a wild-type A2Mpolypeptide. A bait region of a variant A2M polypeptide can have one ormore mutant amino acid regions that are different than those regions ina wild-type A2M polypeptide. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moremutant amino acid regions that are different than those regions in awild-type A2M polypeptide. A mutant bait region of a variant A2Mpolypeptide can replace or substitute a bait region in a wild-type A2Mpolypeptide. A mutant bait region of a variant A2M polypeptide can beany of SEQ ID NOs: 5-66.

The A2M variant polypeptides provided herein also include A2M variantproteins characterized by amino acid sequences similar to those ofpurified A2M variant. Isolated or purified variant A2M polypeptides canhave one or more amino acid residues within the polypeptide that aresubstituted by another amino acid of a similar polarity that acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence can be selected from other members ofthe class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. The aromatic aminoacids include phenylalanine, tryptophan, and tyrosine.

A bait region of a variant A2M polypeptide can have one or more baitregion isoforms. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regionisoforms. A bait region of a variant A2M polypeptide can have one ormore mutant or engineered bait regions. For example, a bait region of avariant A2M polypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or10 or more mutant or engineered bait regions.

A bait region of a variant A2M polypeptide can have one or more copiesof one or more bait regions. The one or more bait regions can be thesame bait regions (repeats), different bait regions, or any combinationthereof. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more copies of one ormore bait regions, wherein the one or more bait regions can be the samebait regions (repeats), different bait regions, or any combinationthereof.

A variant A2M polypeptide can comprise one or more bait regions derivedfrom different organisms, different species of an organism, or acombination thereof. For example, a variant A2M polypeptide can have 2or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regions derived fromdifferent organisms, different species of an organism, or a combinationthereof. One or more bait regions derived from different organisms canbe derived from one or more different organisms and not from differentspecies of an organism. For example, one or more modified bait regionscan be derived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moredifferent organisms and not contain 2 or more bait regions derived fromdifferent species of an organism. One or more bait regions derived fromdifferent species of an organism can be derived from one or moredifferent species of an organism and not from different organisms. Forexample, one or more modified bait regions can be derived from 2 ormore, or 3, 4, 5, 6, 7, 8, 9, or 10 or more different species of anorganism and not contain 2 or more bait regions derived from differentorganism. The modified bait regions can be derived from any animal,insect, plant, bacteria, viral, yeast, fish, reptile, amphibian, orfungi. The modified bait regions can be derived from any animal with A2Mor homologous protein, such as pig, mouse, rat, rabbit, cat, dog, frog,monkey, horse or goat.

A variant A2M polypeptide can comprise one or more bait regions ofvariant A2M polypeptides. For example, a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait region ofvariant A2M polypeptides. One or more bait region of a variant A2Mpolypeptides can be derived from one or more different species. Forexample, one or more bait regions of variant A2M polypeptides can bederived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more differentspecies. The bait region of variant A2M polypeptides can be derived fromany animal, insect, plant, bacteria, viral, yeast, fish, reptile,amphibian, or fungi species.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more proteins other than A2M.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from A2M.A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more non-natural protein sequences. The non-natural protein sequencescan comprise one or more protease recognition sites in series and canfunction as bait for proteases. A variant A2M polypeptide can have aplurality of protease recognition sites that can be one or more proteasesubstrate bait regions from or any of the combination of bait regionsdescribed herein. A variant A2M polypeptide can have any number ofprotease bait regions arranged in series. A variant A2M polypeptide canhave any number of protease bait regions from any species and can bearranged in series. One or more protease substrate bait regions from oneor more proteins other than A2M or from the one or more non-naturalprotein sequences can be a suicide inhibitor. For example, a variant A2Mpolypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore suicide inhibitor bait regions. A suicide inhibitor can be operableto covalently attach a protease to A2M. Examples of known recognitionsequences for exemplary ADAMTSs and MMPs in human aggrecan are indicatedin Table 1. Dash shows location of proteolysis.

TABLE 1 Protease Aggrecan Cleavage Site Sequence ADAMTSs370NITEGE-ARGS377 ADAMTSs 1540TASELE-GRGTI1550 ADAMTSs1709TFKEEE-GLGSV1719 MMP-8 370NITEGE-ARGS377 MMPs 336VDIPEN-FFG344 MMP-3374ARGS-V378 MMP-13 379ILTVKP-IFEV388

A variant A2M polypeptide can be characterized by at least about a 10%increase in protease inhibitory effectiveness compared to the proteaseinhibitory effectiveness of a wild type A2M protein. For example, avariant A2M polypeptide can be characterized by at least about a 20,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% increase in protease inhibitory effectiveness when comparedto the protease inhibitory effectiveness of a wild type A2M protein. Avariant A2M polypeptide can be characterized by an increase in proteaseinhibitory effectiveness compared to the protease inhibitoryeffectiveness of a wild type A2M protein. For example, a variant A2Mpolypeptide can be characterized by an 1.2, 1.2, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times increase in protease inhibitoryeffectiveness compared to the protease inhibitory effectiveness of awild type A2M protein.

A variant A2M polypeptide can be characterized as having an increasedability to inhibit one or more proteases compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to inhibitone or more proteases that is at least 1.5 times higher than the abilityof a wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times higher than theability of a wild-type A2M polypeptide to inhibit the one or moreproteases. A variant A2M polypeptide can have an ability to inhibit oneor more proteases that is from 1.5-100 times higher than the ability ofa wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is from 1.6-100, 1.7-100, 1.8-100, 1.9-100, 2-100,2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100, 2.6-100, 2.7-100, 2.8-100,2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100, 3.4-100, 3.5-100, 3.6-100,3.7-100, 3.8-100, 3.9-100, 4-100, 5-100, 6-100, 7-100, 8-100, 9-100,10-100, 11-100, 12-100, 13-100, 14-100, 15-100, 16-100, 17-100, 18-100,19-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 60-100,70-100, 80-100, 90-100, 1.5-90, 1.6-90, 1.7-90, 1.8-90, 1.9-90, 2-90,2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90, 2.6-90, 2.7-90, 2.8-90, 2.9-90,3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90, 3.5-90, 3.6-90, 3.7-90, 3.8-90,3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90, 9-90, 10-90, 11-90, 12-90, 13-90,14-90, 15-90, 16-90, 17-90, 18-90, 19-90, 20-90, 25-90, 30-90, 35-90,40-90, 45-90, 50-90, 60-90, 70-90, 80-90, 1.5-80, 1.6-80, 1.7-80,1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80, 2.3-80, 2.4-80, 2.5-80, 2.6-80,2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80, 3.2-80, 3.3-80, 3.4-80, 3.5-80,3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80, 5-80, 6-80, 7-80, 8-80, 9-80,10-80, 11-80, 12-80, 13-80, 14-80, 15-80, 16-80, 17-80, 18-80, 19-80,20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 60-80, 70-80, 1.5-70,1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70, 2.1-70, 2.2-70, 2.3-70, 2.4-70,2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70, 3.0-70, 3.1-70, 3.2-70, 3.3-70,3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70, 3.9-70, 4-70, 5-70, 6-70, 7-70,8-70, 9-70, 10-70, 11-70, 12-70, 13-70, 14-70, 15-70, 16-70, 17-70,18-70, 19-70, 20-70, 25-70, 30-70, 35-70, 40-70, 45-70, 50-70, 60-70,1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60, 2-60, 2.1-60, 2.2-60, 2.3-60,2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60, 2.9-60, 3.0-60, 3.1-60, 3.2-60,3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60, 3.8-60, 3.9-60, 4-60, 5-60,6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60, 13-60, 14-60, 15-60, 16-60,17-60, 18-60, 19-60, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60,1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50, 2-50, 2.1-50, 2.2-50, 2.3-50,2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50, 2.9-50, 3.0-50, 3.1-50, 3.2-50,3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50, 3.8-50, 3.9-50, 4-50, 5-50,6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50, 13-50, 14-50, 15-50, 16-50,17-50, 18-50, 19-50, 20-50, 25-50, 30-50, 35-50, 40-50, 1.5-40, 1.6-40,1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40, 2.2-40, 2.3-40, 2.4-40, 2.5-40,2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40, 3.1-40, 3.2-40, 3.3-40, 3.4-40,3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40, 4-40, 5-40, 6-40, 7-40, 8-40,9-40, 10-40, 11-40, 12-40, 13-40, 14-40, 15-40, 16-40, 17-40, 18-40,19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30, 1.7-30, 1.8-30, 1.9-30,2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30, 2.6-30, 2.7-30, 2.8-30,2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30, 3.5-30, 3.6-30, 3.7-30,3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30,13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 1.5-20, 1.6-20,1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20, 2.2-20, 2.3-20, 2.4-20, 2.5-20,2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20, 3.1-20, 3.2-20, 3.3-20, 3.4-20,3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20, 4-20, 5-20, 6-20, 7-20, 8-20,9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 1.5-15, 1.6-15, 1.7-15,1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15, 2.3-15, 2.4-15, 2.5-15, 2.6-15,2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15, 3.2-15, 3.3-15, 3.4-15, 3.5-15,3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15,10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10, 1.6-10, 1.7-10, 1.8-10,1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10, 2.5-10, 2.6-10, 2.7-10,2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10, 3.4-10, 3.5-10, 3.6-10,3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1.5-9,1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9, 2.4-9, 2.5-9,2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9, 3.4-9, 3.5-9,3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1.5-8, 1.6-8,1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8, 2.5-8, 2.6-8,2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8, 3.5-8, 3.6-8,3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7, 1.7-7, 1.8-7,1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7, 2.7-7, 2.8-7,2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7, 3.7-7, 3.8-7,3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6, 2-6, 2.1-6,2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6, 3.0-6, 3.1-6,3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6, 4-6, 5-6, 1.5-5,1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5, 2.4-5, 2.5-5,2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5, 3.4-5, 3.5-5,3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4, 1.8-4, 1.9-4, 2-4,2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4, 2.8-4, 2.9-4, 3.0-4,3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4, 3.8-4, 3.9-4, 1.5-3,1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3, 2.3-3, 2.4-3, 2.5-3,2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2, 1.8-2, or 1.9-2 timeshigher than the ability of a wild-type A2M polypeptide to inhibit theone or more proteases.

The one or more proteases can include a matrix metalloprotease, such asMMP1 (Interstitial collagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin1), MMP7 (Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11), Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP2? (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease, such as ADAMTS1, ADAMTS2, ADAMTS3,ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (METH-2),ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16,ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin; elastase;compliment factors; dotting factors; thrombin; plasmin; subtilisin;Neprilysin; Procollagen peptidase; Thermolysin; Pregnancy-associatedplasma protein A; Bone morphogenetic protein 1; Lysostaphin; Insulindegrading enzyme; ZMPSTE2; and acetylcholinesterase.

A variant A2M polypeptide can be characterized as having an increasedability to prevent FAC formation compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to preventFAC formation that is at least 1.5 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. For example, avariant A2M polypeptide can have an ability to prevent FAC formationthat is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, or 100 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. A variant A2Mpolypeptide can have an ability to prevent FAC formation that is from1.5-100 times higher than the ability of a wild-type A2M polypeptide toprevent FAC formation. For example, a variant A2M polypeptide can havean ability to prevent FAC formation that is from 1.6-100, 1.7-100,1.8-100, 1.9-100, 2-100, 2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100,2.6-100, 2.7-100, 2.8-100, 2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100,3.4-100, 3.5-100, 3.6-100, 3.7-100, 3.8-100, 3.9-100, 4-100, 5-100,6-100, 7-100, 8-100, 9-100, 10-100, 11-100, 12-100, 13-100, 14-100,15-100, 16-100, 17-100, 18-100, 19-100, 20-100, 25-100, 30-100, 35-100,40-100, 45-100, 50-100, 60-100, 70-100, 80-100, 90-100, 1.5-90, 1.6-90,1.7-90, 1.8-90, 1.9-90, 2-90, 2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90,2.6-90, 2.7-90, 2.8-90, 2.9-90, 3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90,3.5-90, 3.6-90, 3.7-90, 3.8-90, 3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90,9-90, 10-90, 11-90, 12-90, 13-90, 14-90, 15-90, 16-90, 17-90, 18-90,19-90, 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 60-90, 70-90,80-90, 1.5-80, 1.6-80, 1.7-80, 1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80,2.3-80, 2.4-80, 2.5-80, 2.6-80, 2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80,3.2-80, 3.3-80, 3.4-80, 3.5-80, 3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80,5-80, 6-80, 7-80, 8-80, 9-80, 10-80, 11-80, 12-80, 13-80, 14-80, 15-80,16-80, 17-80, 18-80, 19-80, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80,50-80, 60-80, 70-80, 1.5-70, 1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70,2.1-70, 2.2-70, 2.3-70, 2.4-70, 2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70,3.0-70, 3.1-70, 3.2-70, 3.3-70, 3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70,3.9-70, 4-70, 5-70, 6-70, 7-70, 8-70, 9-70, 10-70, 11-70, 12-70, 13-70,14-70, 15-70, 16-70, 17-70, 18-70, 19-70, 20-70, 25-70, 30-70, 35-70,40-70, 45-70, 50-70, 60-70, 1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60,2-60, 2.1-60, 2.2-60, 2.3-60, 2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60,2.9-60, 3.0-60, 3.1-60, 3.2-60, 3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60,3.8-60, 3.9-60, 4-60, 5-60, 6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60,13-60, 14-60, 15-60, 16-60, 17-60, 18-60, 19-60, 20-60, 25-60, 30-60,35-60, 40-60, 45-60, 50-60, 1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50,2-50, 2.1-50, 2.2-50, 2.3-50, 2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50,2.9-50, 3.0-50, 3.1-50, 3.2-50, 3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50,3.8-50, 3.9-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50,13-50, 14-50, 15-50, 16-50, 17-50, 18-50, 19-50, 20-50, 25-50, 30-50,35-50, 40-50, 1.5-40, 1.6-40, 1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40,2.2-40, 2.3-40, 2.4-40, 2.5-40, 2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40,3.1-40, 3.2-40, 3.3-40, 3.4-40, 3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40,4-40, 5-40, 6-40, 7-40, 8-40, 9-40, 10-40, 11-40, 12-40, 13-40, 14-40,15-40, 16-40, 17-40, 18-40, 19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30,1.7-30, 1.8-30, 1.9-30, 2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30,2.6-30, 2.7-30, 2.8-30, 2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30,3.5-30, 3.6-30, 3.7-30, 3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30,9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30,19-30, 20-30, 1.5-20, 1.6-20, 1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20,2.2-20, 2.3-20, 2.4-20, 2.5-20, 2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20,3.1-20, 3.2-20, 3.3-20, 3.4-20, 3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20,4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20,15-20, 1.5-15, 1.6-15, 1.7-15, 1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15,2.3-15, 2.4-15, 2.5-15, 2.6-15, 2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15,3.2-15, 3.3-15, 3.4-15, 3.5-15, 3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15,5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10,1.6-10, 1.7-10, 1.8-10, 1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10,2.5-10, 2.6-10, 2.7-10, 2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10,3.4-10, 3.5-10, 3.6-10, 3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10,8-10, 9-10, 1.5-9, 1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9,2.4-9, 2.5-9, 2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9,3.4-9, 3.5-9, 3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9,1.5-8, 1.6-8, 1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8,2.5-8, 2.6-8, 2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8,3.5-8, 3.6-8, 3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7,1.7-7, 1.8-7, 1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7,2.7-7, 2.8-7, 2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7,3.7-7, 3.8-7, 3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6,2-6, 2.1-6, 2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6,3.0-6, 3.1-6, 3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6,4-6, 5-6, 1.5-5, 1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5,2.4-5, 2.5-5, 2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5,3.4-5, 3.5-5, 3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4,1.8-4, 1.9-4, 2-4, 2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4,2.8-4, 2.9-4, 3.0-4, 3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4,3.8-4, 3.9-4, 1.5-3, 1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3,2.3-3, 2.4-3, 2.5-3, 2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2,1.8-2, or 1.9-2 times higher than the ability of a wild-type A2Mpolypeptide to prevent FAC formation.

One aspect of the invention is a method for determining the enhancedinhibition of a protease by a variant A2M polypeptide comprising: a)providing a variant A2M polypeptide comprising a sequence of one or moreof SEQ ID NOs 5-66; b) contacting the variant A2M polypeptide with theprotease and a substrate cleaved by the protease; c) contacting awild-type A2M polypeptide with the protease and the substrate cleaved bythe protease; and d) comparing the amount of cleavage of the substratefrom step b) to the amount of cleavage of the substrate from step c),thereby determining the enhanced inhibition of the protease by thevariant A2M polypeptide.

Enzymatic glycoconjugation reactions can be targeted to glycosylationsites and to residues that are attached to glycosylation sites. Thetargeted glycosylation sites can be sites native to a wild-type A2Mprotein, native to a variant A2M polypeptide or, alternatively, they canbe introduced into a wild-type A2M or variant A2M polypeptide bymutation. Thus, a method for increasing the in vivo half life of avariant A2M polypeptide is provided by the methods of the invention.

A variant A2M polypeptide can include an amino acid sequence thatmutated to insert, remove or relocate one or more glycosylation site inthe protein. When a site is added or relocated, it is not present or notpresent in a selected location in the wild-type A2M peptide. The mutantglycosylation site can be a point of attachment for a modified glycosylresidue that can be enzymatically conjugated to the glycosylation site.Using the methods of the invention, the glycosylation site can beshifted to any efficacious position on the peptide. For example, if thenative glycosylation site is sufficiently proximate or within the baitregion of variant A2M polypeptide peptide that conjugation interfereswith the ability to bind a protease, inhibit a protease, or acombination thereof, it is within the scope of the invention to engineera variant A2M polypeptide that includes a glycosylation site as modifiedor removed from the bait as necessary to provide a biologically activevariant A2M polypeptide.

Any glycosyltransferase or method of their use known in the art can beused for in vitro enzymatic synthesis of variant A2M polypeptides withcustom designed glycosylation patterns, various glycosyl structures, ora combination thereof possible. See, for example, U.S. Pat. Nos.5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826;US2003180835; and WO 03/031464.

The present invention provides methods of improving or lengthening thein vivo half-lives of variant A2M polypeptides by conjugating awater-soluble polymer to the variant A2M polypeptides through an intactglycosyl linking group. In an exemplary embodiment, covalent attachmentof polymers, such as polyethylene glycol (PEG), to such variant A2Mpolypeptides affords variant A2M polypeptides having in vivo residencetimes, and pharmacokinetic and pharmacodynamic properties, enhancedrelative to the unconjugated variant A2M polypeptide.

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein. The polymer backbone can be linear or branched. Branchedpolymer backbones are generally known in the art. Typically, a branchedpolymer has a central branch core moiety and a plurality of linearpolymer chains linked to the central branch core. PEG is commonly usedin branched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(n) in which R represents the core moiety,such as glycerol or pentaerythritol, and n represents the number ofarms. Many other polymers are also suitable for the invention. Examplesof suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol and the like,poly(oxyethylated polyol), poly(olefinic alcohol),polyvinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine) and copolymers, terpolymers, and mixturesthereof. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 100 Da toabout 100,000 Da often from about 6,000 Da to about 80,000 Da.

A variant A2M polypeptide can further comprise PEG. A variant A2Mpolypeptide can have one or more mutant or modified glycosylation sites.The modified glycosylation sites can comprise PEG. For example, avariant A2M polypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more mutant or modified glycosylation sites. The conjugationor addition of PEG to a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites can result in a variant A2Mpolypeptide with a longer half life than the half life of a wild-typeA2M protein without PEG when disposed within a subject, such as a jointor spine disc of a subject. The conjugation or addition of PEG to avariant A2M polypeptide with one or more modified or abnormalglycosylation sites can result in a variant A2M polypeptide with alonger half life than the half life of a variant A2M polypeptide withoutone or more modified glycosylation sites without PEG when disposedwithin a subject, such as a joint or spine disc of a subject. Theconjugation or addition of PEG to a variant A2M polypeptide with one ormore modified or abnormal glycosylation sites can result in a variantA2M polypeptide with a longer half life than the half life of a variantA2M polypeptide with one or more modified glycosylation sites withoutPEG when disposed within a subject, such as a joint or spine disc of asubject. For example, a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites with PEG can have half lifethat is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the half life of awild type A2M protein without PEG, a variant A2M polypeptide with one ormore modified glycosylation sites without PEG, or a variant A2Mpolypeptide without one or more modified glycosylation sites withoutPEG. For example, a variant A2M polypeptide with one or more modified orabnormal glycosylation sites with PEG can have half life that is 2 timesthe half life of a wild type A2M protein composition with one or moremodified or abnormal glycosylation sites without PEG when disposedwithin a joint or spine disc of a subject.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” can be intended nucleotide fragments which differfrom a nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

Fragments of the A2M variants of the present invention which are capableof exhibiting biological activity are also encompassed by the presentinvention. Fragments of the A2M variants can be in linear form or theycan be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments can be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. The presentinvention also provides both full-length and mature forms (for example,without a signal sequence or precursor sequence) of the disclosed A2Mvariants. The protein coding sequence can be identified in the sequencelisting by translation of the disclosed nucleotide sequences. The matureform of such A2M variants can be obtained by expression of a full-lengthpolynucleotide in a suitable mammalian cell or other host cell. Thesequence of the mature form of the A2M variants can be also determinablefrom the amino acid sequence of the full-length form. Where A2M variantsof the present invention are membrane bound, soluble forms of the A2Mvariants are also provided. In such forms, part or all of the regionscausing the A2M variants to be membrane bound are deleted so that theA2M variants are fully secreted from the cell in which it can beexpressed. A2M variant compositions of the present invention can furthercomprise an acceptable carrier, such as a hydrophilic, e.g.,pharmaceutically acceptable, carrier.

Variant A2M Polynucleotide Compositions

As used herein, “A2M polynucleotide,” when used with reference to SEQ IDNOs: 1 or 2, means the polynucleotide sequence of SEQ ID NO: 1 or 2, orfragments thereof, as well as any nucleic acid variants which includeone or more insertions, deletions, mutations, or a combination thereof.The insertions, deletions, and mutations are preferably within thepolynucleotide sequence encoding the bait region of the A2M protein.Similarly, “A2M cDNA”, “A2M coding sequence” or “A2M coding nucleicacid”, when used with reference to SEQ ID NOs: 1 or 2, means the nucleicacid sequences of SEQ ID NOs: 1 or 2, or fragments thereof, as well asnucleic acid variants which include one or more mutations, insertions,deletions, or a combination thereof. The A2M polynucleotides, orfragments thereof, can be manipulated using conventional techniques inmolecular biology so as to create variant A2M recombinant polynucleotideconstructs, encoding the variant A2M polypeptides that express variantA2M polypeptides. Variant A2M polynucleotides include nucleotidesequences having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NOs: 1 and2. A2M coding sequences includes nucleotide sequences having at least99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or85% sequence identity to any one of SEQ ID NOs: 1 and 2.

In one aspect, provided herein is a variant A2M polynucleotidenucleotide composition. Numerous polynucleotide sequences encodingwild-type A2M proteins from various organisms have been determined. AnyA2M DNA sequence identified can be subsequently obtained by chemicalsynthesis and/or a polymerase chain reaction (PCR) technique such asoverlap extension method. For a short sequence, completely de novosynthesis may be sufficient; whereas further isolation of full lengthcoding sequence from a human cDNA or genomic library using a syntheticprobe may be necessary to obtain a larger gene. Alternatively, a nucleicacid sequence encoding an A2M polypeptide can be isolated from a humancDNA or genomic DNA library using standard cloning techniques such aspolymerase chain reaction (PCR), where homology-based primers can oftenbe derived from a known nucleic acid sequence encoding an A2Mpolypeptide.

cDNA libraries suitable for obtaining a coding sequence for a wild-typeA2M polypeptide can be obtained commercially or can be constructed. Thegeneral methods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known. Uponobtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type A2M protein from the cDNAlibrary. A similar procedure can be followed to obtain a full lengthsequence encoding a wild-type A2M protein from a human genomic library.Human genomic libraries are commercially available or can be constructedaccording to various art-recognized methods. In general, to construct agenomic library, the DNA is first extracted from a tissue where apeptide is likely found. The DNA is then either mechanically sheared orenzymatically digested to yield fragments of about 12-20 kb in length.The fragments are subsequently separated by gradient centrifugation frompolynucleotide fragments of undesired sizes and are inserted inbacteriophage λ, vectors. These vectors and phages are packaged invitro. Recombinant phages are analyzed by plaque hybridization.

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type A2M protein can be obtained

Upon acquiring a nucleic acid sequence encoding a wild-type A2M protein,the coding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type A2M protein can beexpressed mutated into a variant A2M polypeptide of the inventionproduced from the resulting construct. Further modifications to thewild-type A2M protein coding sequence, for example, nucleotidesubstitutions, may be subsequently made to alter the bait region of theA2M protein.

The present invention further provides isolated polypeptides encoded bythe polynucleotides, or fragments thereof, of the present invention orby degenerate variants of the polynucleotides, or fragments thereof, ofthe present invention. Preferred polynucleotides, or fragments thereof,of the present invention are the ORFs that encode A2M variants.

A variant A2M polynucleotide can be made by mutating the polynucleotidesequence encoding a wild-type A2M protein. This can be achieved by usingany known mutagenesis methods. Exemplary modifications to a wild-typeA2M polynucleotide for accepting variant bait regions described hereininclude those in SEQ ID NO 2. Exemplary modifications to an A2Mnucleotide include inserting or substituting a nucleotide sequenceencoding a variant bait region of SEQ ID NO 5-66 into the wild-type A2Mpolynucleotide sequence of SEQ ID NO: 1 and the variant A2M acceptorpolynucleotide sequence of SEQ ID NO 2. Mutagenesis procedures can beused separately or in combination to produce variants of a set ofnucleic acids, and hence variants of encoded polypeptides. Kits formutagenesis are commercially available.

In one aspect, provided herein are methods of making any of the variantA2M polynucleotides. A method of making a variant A2M polynucleotide cancomprise inserting or substituting a variant bait region into awild-type A2M polynucleotide sequence or substantially similar sequence.The substantially similar sequence can be SEQ ID NO 2. One aspect of theinvention is a method for making a variant A2M polynucleotidecomprising: a) providing a vector containing a variant A2Mpolynucleotide comprising a sequence of SEQ ID NO 2; b) digesting thevector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; c) ligating one end of the one ormore polynucleotides encoding one or more of the non-natural baitregions of SEQ ID NOs 5-66 to one end of the linear vector; and d)ligating the other end of the one or more polynucleotides encoding oneor more of the non-natural bait regions of SEQ ID NOs 5-66 to the otherend of the linear vector, thereby forming a vector containing a variantA2M polynucleotide comprising the non-natural bait regions of SEQ ID NOs5-66.

Protein Production

A variety of methodologies known in the art can be utilized to obtainany one of the isolated A2M variant proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. Such polypeptides can besynthesized with or without a methionine on the amino terminus.Chemically synthesized polypeptides can be oxidized using methods setforth in these references to form disulfide bridges. The syntheticallyconstructed A2M variant sequences, by virtue of sharing primary,secondary or tertiary structural and/or conformational characteristicswith A2M variants can possess biological properties in common therewith,including protease inhibitory activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the A2M variants. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedA2M variants in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The A2M variant polypeptides of the present invention can alternativelybe purified from cells which have been altered to express the desiredA2M variant. As used herein, a cell can be said to be altered to expressa desired A2M variant polypeptide or protein when the cell, throughgenetic manipulation, can be made to produce a A2M variant polypeptidewhich it normally does not produce or which the cell normally producesat a lower level. One skilled in the art can readily adapt proceduresfor introducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the A2M variant polypeptides of the present invention.

A variant A2M polypeptide can be a recombinant protein, or fragmentsthereof, and can be produced in a host cell or in vitro system.Recombinant polypeptides and protein promoters can be inserted in such amanner that it can be operatively produced in a host cell, for example,a bacterial culture or lower eukaryotes such as yeast or insects or inprokaryotes or any host know in the art. A variant A2M recombinantprotein can be produced in a bacterium, yeast, fungi, insect, ormammalian host cell, or a cell free system. For example, a variant A2Mpolypeptide can be produced in Escherichia coli, Bacillus subtilis,Salmonella typhimurium, Corynebacterium, Saccharomyces cerevisiae,Schizosaccharomyces pombe Kluyveromyces strains, Candida, Pichiapastoris, baculovirus-infected insect cells, or mammalian cells such asCOS cells, BHK cells, 293 cells, 3T3 cells, NSO hybridoma cells, babyhamster kidney (BHK) cells, PER.C6™ human cells, HEK293 cells orCricetulus griseus (CHO) cells. A variant A2M polypeptide can beproduced by transient expression, stable cell lines, BacMam-mediatedtransient transduction, or cell-free protein production.

The variant A2M polypeptides can also be produced by operably linkingthe isolated variant A2M polynucleotides to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the variant A2M nucleotide sequence of interest can be ligatedto an adenovirus transcription/translation control complex, for example,the late promoter and tripartite leader sequence. This chimeric gene canthen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genomecan result in a recombinant virus that is viable and capable ofexpressing the variant A2M gene product in infected hosts. Specificinitiation signals can also be required for efficient translation ofinserted nucleotide sequences. These signals include the ATG initiationcodon and adjacent sequences. In cases where an entire variant A2M geneor cDNA, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, for example, a pJ608mammalian expression vector (FIG. 23) no additional translationalcontrol signals are needed. Exogenous translational control signals,such as the ATG initiation codon, can be provided.

Host cells can be genetically engineered to contain the variant A2Mpolynucleotides of the invention. For example, such host cells cancontain variant A2M polynucleotides introduced into the host cell usingknown transformation, transfection or infection methods. As used herein,a cell capable of expressing a variant A2M polynucleotide can be“transformed.” The variant A2M polypeptides of the invention can beprepared by culturing transformed host cells under culture conditionssuitable to express the recombinant protein. Any procedure forintroducing foreign nucleotide sequences into host cells may be used.Non-limiting examples include the use of calcium phosphate transfection,transfection, DEAE, dextran-mediated transfection, microinjection,lipofection, polybrene, protoplast fusion, electroporation (Davis, L. etal., Basic Methods in Molecular Biology (1986)), liposomes,microinjection, plasma vectors, viral vectors, and any other well knownmethods for introducing cloned genomic DNA, cDNA, synthetic DNA, orother foreign genetic material into a host cell. A genetic engineeringprocedure capable of successfully introducing at least one gene into thehost cell capable of expressing the variant A2M polynucleotide can beused.

The present invention still further provides host cells engineered toexpress the variant A2M polynucleotides of the invention, wherein thevariant A2M polynucleotides are operative with a regulatory sequenceheterologous to the host cell which drives expression of the variant A2Mpolynucleotides in the cell. Knowledge of A2M-like DNA allows formodification of cells to permit, or increase, expression of A2M-likepolypeptide. Cells can be modified, for example, by homologousrecombination, to provide increased variant A2M polypeptide expressionby replacing, in whole or in part, the naturally occurring A2M derivedfrom the SV40 viral genome, for example, SV40 macroglobulin-likepromoter with all or part of a heterologous promoter so that the cells'variant A2M sites can be used to provide the required non-transcribedpolypeptide and can be expressed at higher levels.

For long-term, high-yield production of recombinant variant A2Mpolypeptides, stable expression is preferred. For example, cell linesthat stably express the variant A2M sequences described herein can beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells are allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn are cloned and expandedinto cell lines. This method is advantageously used to engineer celllines which express the variant A2M gene product. Such engineered celllines are particularly useful in screening and evaluation of compoundsthat affect the endogenous activity of the variant A2M gene product. Anumber of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase, and adenine phosphoribosyltransferase genescan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate; gpt,which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G-418; and hygro, which confersresistance to hygromycin.

Variant A2M polynucleotide sequences can be engineered so as to modifyprocessing or expression of the protein. For example, and not by way oflimitation, the variant A2M polynucleotides can be combined with apromoter sequence and/or ribosome binding site, or a signal sequence canbe inserted upstream of variant A2M polynucleotide sequences to permitsecretion of the variant A2M polypeptide and thereby facilitateharvesting or bioavailability. Additionally, a variant A2Mpolynucleotide can be mutated in vitro or in vivo, to create and/ordestroy translation, initiation, and/or termination sequences, or tocreate variations in coding regions and/or form new restriction sites ordestroy preexisting ones, or to facilitate further in vitromodification. Any technique for mutagenesis known in the art can beused, including but not limited to, in vitro site-directed mutagenesis.

Further, nucleic acids encoding other proteins or domains of otherproteins can be joined to nucleic acids encoding variant A2Mpolypeptides or fragments thereof so as to create a fusion protein.Nucleotides encoding fusion proteins can include, but are not limitedto, a full length variant or wild-type A2M protein, a truncated variantor wild-type A2M protein or a peptide fragment of a variant or wild typeA2M protein fused to an unrelated protein or peptide, such as forexample, a transmembrane sequence, which anchors the A2M peptidefragment to the cell membrane; an Ig Fc domain which increases thestability and half life of the resulting fusion protein; maltose bindingprotein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), aHis tag, an enzyme, fluorescent protein, luminescent protein which canbe used as a marker, for example, an A2M-Green Fluorescent Proteinfusion protein. The fusion proteins can be used for affinitypurification.

The variant A2M nucleic acids and polypeptides can also be expressed inorganisms so as to create a transgenic organism. Desirable transgenicplant systems having one or more of these sequences include Arabadopsis,Maize, and Chlamydomonas. Desirable insect systems having one or more ofthe variant A2M polynucleotides and/or polypeptides include, forexample, D. melanogaster and C. elegans. Animals of any species,including, but not limited to, amphibians, reptiles, birds, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees can be used togenerate variant A2M containing transgenic animals. Transgenic organismsdesirably exhibit germline transfer of variant A2M nucleic acids andpolypeptides described herein.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins can possess biological properties incommon therewith, including protein activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies. The polypeptides andproteins of the present invention can alternatively be purified fromcells which have been altered to express the desired polypeptide orprotein. As used herein, a cell can be said to be altered to express adesired polypeptide or protein when the cell, through geneticmanipulation, can be made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells in a suitable culture medium,and purifying the protein from the cells or the culture in which thecells are grown. For example, the methods can include a process forproducing a polypeptide in which a host cell containing a suitableexpression vector that includes a polynucleotide of the invention can becultured under conditions that allow expression of the encodedpolypeptide. The polypeptide can be recovered from the culture,conveniently from the culture medium, or from a lysate prepared from thehost cells and further purified. Preferred embodiments include those inwhich the protein produced by such process can be a full length ormature form of the protein, such as A2M. In an alternative method, thepolypeptide or protein can be purified from bacterial cells whichnaturally produce the polypeptide or protein. One skilled in the art canreadily follow known methods for isolating polypeptides and proteins inorder to obtain one of the isolated polypeptides or proteins of thepresent invention. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography, and immuno-affinity chromatography. See, e.g., Scopes,Protein Purification: Principles and Practice, Springer-Verlag (1994);Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel etal., Current Protocols in Molecular Biology. Polypeptide fragments thatretain biological or immunological activity include fragments comprisinggreater than about 100 amino acids, or greater than about 200 aminoacids, and fragments that encode specific protein domains. The purifiedpolypeptides can be used in in vitro binding assays which are well knownin the art to identify molecules which bind to the polypeptides. Thesemolecules include but are not limited to, for example, small molecules,molecules from combinatorial libraries, antibodies or other proteins.The molecules identified in a binding assay can then be tested forantagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules can betitrated into a plurality of cell cultures or animals and then testedfor either cell or animal death or prolonged survival of the animal orcells.

The resulting expressed variant A2M polypeptides can then be purifiedfrom a culture, for example, from culture medium or cell extracts, usingknown purification processes, such as affinity chromatography, gelfiltration, and ion exchange chromatography. The purification of thevariant A2M polypeptides can also include an affinity column containingagents which will bind to the protein; one or more column steps oversuch affinity resins as concanavalin A-agarose, heparin-toyopearl orCibacron blue 3GA Sepharose™; one or more steps involving hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or immunoaffinity chromatography. Alternatively,the protein of the invention can also be expressed in a form which willfacilitate purification. For example, a protein can be expressed as afusion protein, such as those of maltose binding protein (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag.Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAG®”)is commercially available from Kodak (New Haven, Conn.). Finally, one ormore reverse-phase high performance liquid chromatography (RP-HPLC)steps employing hydrophobic RP-HPLC media, for example, silica gelhaving pendant methyl or other aliphatic groups, can be employed tofurther purify the protein. Any combination of the foregoingpurification procedures can also be employed to provide a substantiallyhomogeneous isolated or purified recombinant variant A2M polypeptide.The variant A2M polypeptides purified can be substantially free of othermammalian proteins and can be defined in accordance with the presentinvention as an “isolated protein.”

Agents for Inhibition of FAC Formation

Also provided herein are methods to inhibit the one or more steps of thefibronectin-aggrecan complex formation cycle (FACC) in a human with acondition or disease (FIG. 1). An agent can be administered to a subjectwith a condition or disease. An agent can be wild-type A2M protein or acomposition described herein, such as a purified form of A2M, or an A2Menriched sample, or a variant A2M polypeptide as described herein. Anagent can be an agent that is not a purified form of A2M concentratedfrom autologous blood. An agent can be an inhibitor or an antagonist. Aninhibitor or antagonist can be a compound or composition that directlyor indirectly, partially or totally blocks activity, decreases,prevents, delays activation, inactivates, desensitizes, or downregulates the activity or expression of a target biomarker. Antagonistscan be, for example, polypeptides, such as antibodies, and solublereceptors, as well as nucleic acids such as siRNA or antisense RNA, aswell as naturally occurring and synthetic biomarker antagonists,including small chemical molecules.

An agent can be compound that has a pharmacological activity. Agents caninclude compositions described herein or compounds that are known drugs,compounds for which pharmacological activity has been identified butthat are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity. An agent can be organic or inorganic chemicalsuch a peptide, protein, including antibodies, small molecules andnatural products.

An agent can comprise an antibody. An antibody can be a polypeptidecomprising a framework region from an immunoglobulin gene or fragmentsthereof that specifically binds and recognizes an antigen. Therecognized immunoglobulin genes can include the kappa, lambda, alpha,gamma, delta, epsilon, and mu constant region genes, as well as themyriad immunoglobulin variable region genes. Light chains can beclassified as either kappa or lambda. Heavy chains can be classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Anantibody can encompass plural referents unless the context clearlyindicates otherwise. In some instances a plurality of the antibodies canbelong to the same antibody species, e.g., in the case of monoclonalantibodies, while in some cases different antibodies species areencompassed the by phrase “an antibody”, e.g., a polyclonal antibodies.An exemplary immunoglobulin (antibody) structural unit can comprise atetramer. Each tetramer can be composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain can definea variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (V,)and variable heavy chain (VH) can refer to these light and heavy chainsrespectively. Antibodies can exist, e.g., as intact immunoglobulins oras a number of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′2, a dimerof Fab which itself is a light chain joined to VH-CH1 by a disulfidebond. The F(ab)′2 may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′2dimer into an Fab′ monomer. The Fab′ monomer can be a Fab with part ofthe hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. An antibody can also be an antibody fragment eitherproduced by the modification of whole antibodies, or those synthesizedde novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty etal., Nature 348:552-554 (1990)). When referring to treatment methods,antibodies that are chimeric, human, humanized or otherwise specific tothe species to be treated can be used.

An agent can be an antibody that binds to the FAC but not to theindividual components of the complex separately. An agent can comprisean antibody that binds to aggrecan or any variation thereof, therebyinhibiting formation of the FAC complex. An agent can comprise anantibody, such as a monoclonal antibody, that binds to aggrecan G3lectin domain. The antibody can bind to aggrecan G3 and prevent theformation of FAC and inflammation. An agent can comprise an antibodythat binds to fibronectin or any variant thereof, thereby inhibitingformation of the FAC complex. An agent can comprise an antibody thatbinds to a PAMP receptor recognition domain of aggrecan, a DAMP receptorrecognition domain of aggrecan, or both, thereby inhibiting activationof monocytes and other cells. Other cells can be macrophages,fibroblast, T-cells, B-cells, neutrophils, platelets, synoviocytes,chondrocytes and other cells involved in inflammation. An agent cancomprise an antibody that binds to a PAMP receptor recognition domain offibronectin, a DAMP receptor recognition domain of fibronectin, or both,thereby inhibiting activation of monocytes and other cells.

An agent that prevents or inhibits FAC formation can be a recombinantaggrecan G3 domain, wherein the domain contains the aggrecan G3 lectindomain and competitively binds to fibronectin; wherein the domain lacksthe Pathogen Associated Molecular Patterns (PAMP) and the DamageAssociated Molecular Patterns (DAMP) receptor recognition domains.Recombinant aggrecan G3 lectin domain can competitively bind tofibronectin. A recombinant aggrecan G3 domain can lack the cellactivation domain and can slow down, inhibit, or prevent FAC formationand inflammation.

An agent that prevents or inhibits FAC formation can be a wild-type A2Mprotein or a recombinant fibronectin fragment, wherein the fragmentcomprises a G3 binding domain and binds to aggrecan, wherein the G3binding domain the PAMP, receptor recognition domain, the DAMP receptorrecognition domain, or both. A recombinant fibronectin fragment cancompetitively bind to aggrecan.

An agent that prevents or inhibits FAC formation can be a soluble formof the PAMP receptor or DAMP receptor that binds to the PAMP domain ofaggrecan G3, the DAMP domain of aggrecan G3, or both, thereby inhibitingactivation of monocytes and other cells to produce proinflammatorycytokines, chemokines, proteases, or any combination thereof.

An agent that prevents or inhibits FAC formation can be a soluble formof the PAMP receptor or DAMP receptor that binds to the PAMP domain offibronectin, the DAMP domain of fibronectin, or both, thereby inhibitingactivation of monocytes and other cells to produce proinflammatorycytokines, chemokines, proteases, or any combination thereof. An agentcan be an inhibitor of fibroblast cells and can inhibit production ofincreased levels fibronectin, recruitment of other fibroblast cells, orboth.

An agent that prevents or inhibits FAC formation can be a smallmolecule. A small molecule can be identified using one or morehigh-throughput screening methods. A small molecule can inhibit FACformation, inhibit activation of monocytes; inhibit increased productionof fibronectin; inhibit recruitment of fibroblast cells; or bind to theDAMP domain of fibronectin, bind to the DAMP domain of aggrecan G3, bindto the PAMP domain of fibronectin, or bind to the PAMP domain ofaggrecan G3, thereby inhibiting activation of cells to produceproinflammatory cytokines, chemokines, proteases, or any combinationthereof. In A small molecule can inhibit FAC formation by competitivelybinding to fibronectin or aggrecan. In some embodiments, the smallmolecule binds to the FAC complex and resulting in dissociation ordegradation of the FAC complex. Inhibiting the formation of thefibronectin-aggrecan complex (FAC) can comprise inhibiting one or moresteps in FAC formation or a step in the FAC formation cycle (FACC).

One or more steps in FAC formation or the FACC can comprise productionof fibronectin in the ECM, production of proteases and metalloproteases,production of inflammatory cytokines and chemokines, degradation ofaggrecan in cartilage, or increasing the aggrecan G3 domain fragmentconcentration.

In any of the methods herein, an agent can be a medicament used to treatjoint injury or inflammation. Thus, one can administer to a subject,along with a composition comprising an elevated concentration of A2M, avariant A2M polypeptide, or a wild-type A2M protein, an effective amountof one or more other medicament (where a composition comprising anelevated concentration of A2M or variant A2M polynucleotide (e.g.,compositions described herein) can be a first medicament). The one ormore other medicaments can include, for example, an immunosuppressiveagent, a cytokine antagonist such as a cytokine antibody, an integrinantagonist (e.g., antibody), a corticosteroid, or any combinationthereof. The type of such second medicament can depend on variousfactors, including the type of inflammation and/or joint damage, theseverity of the inflammation and/or joint damage, the condition and ageof the subject, the type and dose of the first medicament employed, etc.Examples of such additional medicaments include an immunosuppressiveagent (such as mitoxantrone (NOVANTRONE®), MTX, cyclophosphamide,chlorambucil, leflunomide, and azathioprine), intravenous immunoglobulin(gamma globulin), lymphocyte-depleting therapy (e.g., mitoxantrone,cyclophosphamide, CAMPATH™ antibodies, anti-CD4, cladribine, apolypeptide construct with at least two domains comprising ade-immunized, autoreactive antigen or its fragment that can bespecifically recognized by the Ig receptors of autoreactive B-cells (WO2003/68822), total body irradiation, and bone marrow transplantation),integrin antagonist or antibody (e.g., an LFA-1 antibody such asefalizumab/RAPTIVA® commercially available from Genentech, or an alpha 4integrin antibody such as natalizumab/ANTEGREN® available from Biogen,or others as noted above), drugs that treat symptoms secondary orrelated to inflammation and/or joint damage such as those noted herein,steroids such as corticosteroid (e.g., prednisolone, methylprednisolonesuch as SOLU-MEDROL™ methylprednisolone sodium succinate for injection,prednisone such as low-dose prednisone, dexamethasone, orglucocorticoid, e.g., via joint injection, including systemiccorticosteroid therapy), nonlymphocyte-depleting immunosuppressivetherapy (e.g., MMF or cyclosporine), a TNF-α inhibitor such as anantibody to TNF-α or its receptor or TNFR-Ig (e.g., etanercept), DMARD,NSAID, plasmapheresis or plasma exchange, trimethoprim-sulfamethoxazole(BACTRIM™, SEPTRA™), MMF, H2-blockers or proton-pump inhibitors (duringthe use of potentially ulcerogenic immunosuppressive therapy),levothyroxine, cyclosporin A (e.g., SANDIMMUNE®), somatostatin analogue,a DMARD or NSAID, cytokine 25 antagonist such as antibody,anti-metabolite, immunosuppressive agent, rehabilitative surgery,radioiodine, thyroidectomy, anti-IL-6 receptor antagonist/antibody(e.g., ACTEMRA™ (tocilizumab)), or another B-cell antagonist such asBR3-Fc, TACI-Ig, anti-BR3 antibody, anti-CD40 receptor or anti-CD40ligand (CD154), agent blocking CD4O-CD40 ligand, epratuzumab (anti-CD22antibody), lumiliximab (anti-CD23 30 antibody), or anti-CD20 antibodysuch as rituximab or 2H7 antibody. Known inhibitors such as chelators ofknown aggrecanases or MMP's can be administered to a subject in needthereof in amount effective to inhibit or slow down the release ofaggrecan fragments which in effect will reduce or eliminate theformation of the fibronectin aggrecan complexes thereby giving relief tothe subject from the pain.

Diagnostic Methods

Methods for detecting biomarkers, such as a wild-type A2M protein, toidentify sites in the spine or joint that are a source of pain can beused to diagnose, or assist in the diagnosis be of, subjects with painsyndromes related to the anatomic structure and physiologic function ofthe spine or joint. For example, the identification offibronectin-aggrecan complexes in a biological sample, such as abiological sample from the epidural space, intervertebral disc, or facetjoint can be used to diagnose, or assist in the diagnosis be ofradiculopathy, facet joint pain or discogenic pain.

The amount of a biomarker, such as A2M, that can indicate a specificlocation in the spine as a source of pain for a particular subject candepend on numerous factors, including, but not limited to, the age, sex,medical history, etc., of the patient, the site that the biologicalsample was extracted from, and the assay format used to detect thebiomarker. In some embodiments, the level and/or concentration of A2M ina biological sample may be quantified or directly compared with acontrol sample. In some embodiments, the level and/or concentration ofA2M in a biological sample may not be quantified or directly comparedwith a control sample, but can rather be detected relative to a“diagnostic absence” or “diagnostic presence” of A2M.

A “diagnostic absence” can refer to an amount and/or concentration ofA2M in a biological that indicates the absence or likelihood of theabsence of pain or inflammation causing pathology or injury at thelocation from which the sample was taken. A diagnostic absence can bedetectable in a simple assay giving a positive or negative result. Apositive or negative result can be determined based on the amount and/orconcentration of A2M in the biological sample. Detection of a leveland/or concentration of A2M corresponding to a diagnostic absence of A2Mindicates the absence of a pain-causing pathology or injury at thelocation from which the sample was taken. In some embodiments, adiagnostic absence of A2M can be a concentration of A2M in a biologicalsample from about 0-30 μg/ml. For example, a diagnostic absence of A2Mcan be a concentration of A2M in a biological sample from about 0-30μg/ml, 0-25 μg/ml, 0-20 μg/ml, 0-15 μg/ml, 0-10 μg/ml, 0-5 μg/ml, 5-30μg/ml, 5-25 μg/ml, 5-20 μg/ml, 5-15 μg/ml, 5-10 μg/ml, 10-30 μg/ml,10-25 μg/ml, 10-20 μg/ml, 10-15 μg/ml, 15-30 μg/ml, 15-25 μg/ml, 15-20μg/ml, 20-30 μg/ml, or 20-25 μg/ml. In some embodiments, a diagnosticabsence of A2M can be a concentration of A2M in a biological sample fromabout 0-40 μg/ml. For example, a diagnostic absence of A2M can be aconcentration of A2M in a biological sample from about 0-40 μg/ml, 5-40μg/ml, 10-40 μg/ml, 15-40 μg/ml, 20-40 μg/ml, 25-40 μg/ml, 30-40 μg/ml,or 35-40 μg/ml.

In some embodiments, a diagnostic absence of A2M in a biological samplecan be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000 or more fold lower than a controlsample.

A “diagnostic presence” can refer to an amount and/or concentration ofA2M in a biological that indicates the presence or likelihood of thepresence of pain or inflammation causing pathology or injury at thelocation from which the sample was taken. A diagnostic presence can bedetectable in a simple assay giving a positive or negative result. Apositive or negative result can be determined based on the amount and/orconcentration of A2M in the biological sample. Detection of a leveland/or concentration of A2M corresponding to a diagnostic presence ofA2M indicates the presence of a pain-causing pathology or injury at thelocation from which the sample was taken. In some embodiments, adiagnostic presence of A2M can be a concentration of A2M in a biologicalsample of at least about 31 μg/ml, 32 μg/ml, 33 μg/ml, 34 μg/ml, 35μg/ml, 36 μg/ml, 37 μg/ml, 38 μg/ml, or 39 μg/ml. In some embodiments, adiagnostic presence of A2M can be a concentration of A2M in a biologicalsample of at least about 40 μg/ml. For example, a diagnostic presence ofA2M can be a concentration of A2M in a biological sample of at leastabout 45 μg/ml, 50 μg/ml, 55 μg/ml, 60 μg/ml, 65 μg/ml, 70 μg/ml, 75μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, 95 μg/ml, 100 μg/ml, 110 μg/ml, 120μg/ml, 130 μg/ml, 140 μg/ml, 145 μg/ml, 150 μg/ml, 160 μg/ml, 170 μg/ml,180 μg/ml, 190 μg/ml, 200 μg/ml, 220 μg/ml, 240 μg/ml, 250 μg/ml, 260μg/ml, 280 μg/ml, 300 μg/ml, 320 μg/ml, 340 μg/ml, 360 μg/ml, 380 μg/ml,400 μg/ml, 420 μg/ml, 440 μg/ml, 460 μg/ml, 480 μg/ml, 500 μg/ml, ormore.

In some embodiments, a diagnostic presence of A2M can be a concentrationof A2M in a biological sample from about 40-500 μg/ml. For example, adiagnostic presence of A2M can be a concentration of A2M in a biologicalsample from about 50-500 μg/ml, 60-500 μg/ml, 70-500 μg/ml, 80-500μg/ml, 90-500 μg/ml, 100-500 μg/ml, 125-500n/ml, 150-500n/ml, 175-500,200-500n/ml, 250-500n/ml, 300-500 μg/ml, 400-500n/ml, 50-60 μg/ml, 50-70μg/ml, 50-80 μg/ml, 50-90 μg/ml, 50-100 μg/ml, 50-125 μg/ml, 50-150μg/ml, 50-175 μg/ml, 50-200 μg/ml, 50-250 μg/ml, 50-300 μg/ml, 50-400μg/ml, 60-70 μg/ml, 60-80 μg/ml, 60-90 μg/ml, 60-100 μg/ml, 60-125μg/ml, 60-150 μg/ml, 60-175 μg/ml, 60-200 μg/ml, 60-250 μg/ml, 60-300μg/ml, 60-400 μg/ml, 70-80 μg/ml, 70-90 μg/ml, 70-100 μg/ml, 70-125μg/ml, 70-150 μg/ml, 70-175 μg/ml, 70-200 μg/ml, 70-250 μg/ml, 70-300μg/ml, 70-400 μg/ml, 80-90 μg/ml, 80-100 μg/ml, 80-125 μg/ml, 80-150μg/ml, 80-175 μg/ml, 80-200 μg/ml, 80-250 μg/ml, 80-300 μg/ml, 80-400μg/ml, 90-100 μg/ml, 90-125 μg/ml, 90-150 μg/ml, 90-175 μg/ml, 90-200μg/ml, 90-250 μg/ml, 90-300 μg/ml, 90-400 μg/ml, 100-125n/ml,100-150n/ml, 100-175 μg/ml, 100-200n/ml, 100-250n/ml, 100-300 μg/ml,100-400n/ml, 125-150n/ml, 125-175n/ml, 125-200 μg/ml, 125-250n/ml,125-300n/ml, 125-400n/ml, 150-175n/ml, 150-200n/ml, 150-250 μg/ml,150-300n/ml, 150-400n/ml, 175-200n/ml, 175-250n/ml, 175-300n/ml, 175-400μg/ml, 200-250n/ml, 200-300n/ml, 200-400n/ml, 250-300n/ml, 250-400n/ml,or 300-400 μg/ml.

In some embodiments, a diagnostic presence of A2M in a biological samplecan be at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 200, 500, 1000 or more fold higher than a controlsample.

The disclosed methods can be used regardless of whether A2M is normallypresent, or expected to be present, in a particular control sample. Forexample, A2M may not be detectable in certain normal spine samples (suchas, for example, in a disc space or epidural space lavasate) using aparticular assay, resulting in a complete absence of A2M complexes in acontrol biological sample. For example, A2M cannot be detectable incertain normal joint samples (such as, for example, in a synovial fluidsample) using a particular assay, resulting in a complete absence of A2Min a control biological sample. For such biological samples, adiagnostic presence can refer to any detectable amount of A2M using thatsame assay. In other instances, however, there can be a detectable levelof A2M present in normal or control samples and a diagnostic absencerepresents a level that can be lower than the normal level, preferablyrepresenting a statistically significant decrease over the normal level.

Control samples can be samples that are taken from an individual or agroup of individuals not experiencing inflammation or pain, such asspinal or joint pain. Alternatively, control samples can be obtainedfrom a source not suspected to be a source of pain or inflammation, suchas a level of the spine not suspected to be a source of pain. Forexample, in a subject experiencing discogenic pain, the control samplecan be obtained from an unaffected or asymptomatic disc space of thesame patient. Control samples can be samples that are taken from anindividual or a group of individuals not experiencing joint-relatedpain. Alternatively, control samples can be obtained from unaffected orasymptomatic joints from the subject being tested. Particularly suitablejoints to obtain control samples from are joints that are unaffected orasymptomatic contra-lateral to the joint being tested for a diagnosticpresence of A2M. For example, in a subject experiencing left knee pain,the control sample can be obtained from the right knee of the samesubject, provided that the right knee can be unaffected or asymptomatic.

The level of a biomarker, such as A2M, need not be quantified for adiagnostic absence or presence to be detected. Rather, any method ofdetermining whether A2M is present at levels lower or higher than in anormal or control can be used. In addition, a diagnostic absence orpresence does not refer to any absolute quantity of A2M, but rather toan amount that, depending on the biological sample, assay conditions,medical condition of the patient, etc., can be sufficient to distinguishthe level in an affected patient from a normal or control patient.

The presence, absence or level of A2M present at a particular levelwithin the spine can be used to diagnose, or assist in the diagnosis beof, a particular type of spinal pain, such as discogenic, facetogenic orradiculopathic pain. Additionally, or alternatively, the presence,absence, or level of A2M in a spinal sample can be used to distinguishpain that results from spinal pathology or injury from pain originatingfrom another source, such as muscular pain.

The presence, absence, or level of A2M present in a particular joint canbe used to diagnose, or assist in the diagnosis be of, a particular typeof joint-related pain, including, but not limited to, osteoarthritis,meniscal pathology, rotator cuff tears, tendon or ligament pathology,chondrosis, or myofascial pain. In some embodiments, the presence,absence, or level of A2M in a joint can be indicative of pathology orinjury in that particular joint. Additionally, or alternatively, thepresence, absence, or level of A2M in a joint sample can be used todistinguish joint-related pain from pain from another anatomical orphysiological source, such as the spine. For example, the absence or lowlevel of A2M in a joint sample compared to a control sample can be usedto distinguish joint-related pain from radiculopathic pain.

The presence, absence, or change over time in the level of A2M in abiological sample can be used to designate a patient as candidate for aparticular treatment. A spinal sample obtained from the patient can beanalyzed for the presence or absence of A2M. The patient can be selectedfor treatment if A2M is not detected in the spinal sample. The type oftreatment can be then tailored to the severity of the condition asdetermined by the presence, absence, or level of A2M.

The level A2M present at a specific site can also be useful to determinea prognosis for the subject being tested. For example, the level of A2Mpresent in a spinal sample can indicate the extent of an acute injury tothe spine and can assist a practitioner in determining to what extentsuccessful repair or healing of the injury or pathology can be achieved.

Methods for detecting A2M to identify joints as sites for treatingjoint-related pain can be used to diagnose, or assist in the diagnosisof, subjects with pain syndromes related to the anatomic structure andphysiologic function of the synovial joints of the appendicularskeleton. For example, the identification of A2M in a joint can be usedto diagnose, or assist in the diagnosis of osteoarthritis, meniscalpathology, rotator cuff tears, tendon or ligament pathology, chondrosis,or myofascial pain.

Detection of A2M can be used alone, or in combination with otherdiagnostic approaches to diagnose joint-related pain. Exemplarydiagnostic approaches include, but are not limited to, medical historyand physical examination, x-ray radiography, MRI and intra-articularinjection. The presence of A2M can however be used to diagnose injuryand administer treatment at a particular location irrespective ofwhether injury was detectable by other methods, e.g., an MRI. Thepatient will typically be treated by administration of a therapeuticagent to the site of injury or pathology, i.e., the site of presence ofA2M.

The diagnostic methods of the present invention can includedetermination of the expression levels of a set of nucleic acidmolecules comprising polynucleotide sequences coding for a proteinmarker. The diagnostic methods of the present invention can include thedetermination of expression levels of a plurality (i.e., one or more,e.g., at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10 or more) of polypeptides ina biological sample obtained from a subject. Determination of proteinexpression levels in the practice of the inventive methods can beperformed by any suitable method (see, for example, E. Harlow and A.Lane, “Antibodies: A Laboratories Manual”, 1988, Cold Spring HarborLaboratory: Cold Spring Harbor, N.Y.).

The disclosed methods can also be used to assess the efficacy of atreatment or a course of treatment. For example, in a patient with jointpain or radiculopathy testing positive for a diagnostic positive of A2M,such as wild-type A2M protein, indicative or radiculopathy, the efficacyof an anti-inflammatory treatment can be assessed by monitoring, overtime, the levels of A2M. A decrease in the levels of A2M in a biologicalsample taken from a patient following a treatment, compared to a levelin a sample taken from the same patient before, or earlier in, thetreatment, can indicate efficacious treatment. An increase or lack ofchange in the levels of A2M in a biological sample taken from a patientfollowing a treatment, compared to a level in a sample taken from thesame patient before, or earlier in, the treatment, can indicate anon-efficacious treatment.

Inflammation biomarkers for diagnostic methods can be A2M, chemokines,cytokines, fibronectin, or aggrecan polypeptides in any ratio, inaddition to other inflammatory mediators, extracellular matrix moleculesor their breakdown products, signal transduction mediators, proteasesand their inhibitors, and neurotransmitter receptors including, but notlimited to IL-6, Prostaglandin E2, NO, IFN gamma, 5HT, RANTES, MIP-1α,MCP-1, IL-1ra, TNF-α, Procollagens, CTX II, ARGS, aggrecan fragments,fibronectin fragments, FAC, COMP, CS 846, chondroitin fragments, sRAGE,MMP-3, MMP-13 and other MMPs, ADAMTS-4, aggrecanases, NF-kappa-B, p38MAP kinase, DR5/DcR2. The biomarkers can include full lengthpolypeptides or can include fragments of polypeptides.

Any known method for detecting the presence of polypeptides in abiological sample can be used to qualitatively or quantitatively detectthe presence of A2M in biological samples, such as spinal or jointsamples. Suitable methods include, but are not limited to,chromatographic methods, selective binding assays, mass spectrometry,spectrophotometry, or combinations thereof.

Exemplary binding assays include immunoassays, such as enzyme-linkedimmunosorbent assays. Immunoassays can be used to qualitatively orquantitatively analyze a spinal sample for the presence of A2M. Ageneral overview of the applicable technology can be found in a numberof readily available manuals, e.g., Harlow & Lane, Cold Spring HarborLaboratory Press, Using Antibodies: A Laboratory Manual (1999).

The disclosed methods and kits can utilize selective binding partners ofinflammation biomarkers to identify their presence or determine theirlevels in samples from the spine or joint. The selective bindingpartners can be antibodies, or other biomolecules that specifically bindto A2M, or fragments or complexes thereof.

Monoclonal or polyclonal antibodies can be used. The antibodies can beany known in the art, including commercially available antibodies. It iswell known to those of skill in the art that the type, source and otheraspects of an antibody to be used can be a consideration to be made inlight of the assay in which the antibody can be used. In some instances,antibodies that will recognize its antigen target (for instance, anepitope or multiple epitopes from A2M) on a Western blot might not beapplicable to all ELISA or ELISpot assay and vice versa.

Antibodies, antibody fragments, or single chain antibodies to be usedcan be produced using techniques for producing monoclonal or polyclonalantibodies that are well known in the art (see, e.g., Coligan, CurrentProtocols in Immunology (1991); Harlow & Lane, supra; Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986); and Kohler &Milstein, Nature 256: 495-497 (1975). Such techniques include antibodypreparation by selection of antibodies from libraries of recombinantantibodies in phage or similar vectors, as well as preparation ofpolyclonal and monoclonal antibodies by immunizing rabbits or mice (see,e.g. Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature341:544-546 (1989)).

A number of immunogens from A2M can be used to produce antibodiesspecifically reactive with A2M and fragments thereof. For example, arecombinant A2M or an antigenic fragment thereof, can be isolated usingmethods well known to those of skill in the art. Recombinant protein canbe expressed in eukaryotic or prokaryotic cells. Recombinant protein canbe the typically used immunogen for the production of monoclonal orpolyclonal antibodies. Alternatively, synthetic peptides derived fromthe known sequences A2M and conjugated to a carrier protein can be usedas an immunogen. Naturally-occurring protein can also be used either inpure or impure form. The product can be then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated, for subsequent use in immunoassays tomeasure the protein.

Antibodies that specifically bind to complexes containing A2M can beused as specific binding partners.

Non-antibody polypeptides can be used as specific binding agents for thedetection of A2M, or fragments or complexes thereof. A large number ofproteins that specifically bind to A2M are known in the art. Exemplaryproteins that can be used as selective binding partners of A2M include,but are not limited to soluble receptors, cytokines and growth factorsthat are known to bind A2M, modified proteases that can bind to A2M andnot trigger the conformation change.

Once selective binding partners are available, each specific biomarkercan be detected by a variety of selective binding assays, includingimmunoassay methods. For a review of immunological and immunoassayprocedures, see Basic and Clinical Immunology (Stites & Ten eds., 7thed. 1991). Moreover, the disclosed selective binding assays can beperformed in any of several configurations. Several immunoassayconfigurations are reviewed extensively in Enzyme Immunoassay (Maggio,ed., 1980).

Methods for detecting the presence and/or measuring a level of A2M in aspinal or joint sample, can use specific binding partners A2M, orfragments or complexes thereof. The methods generally include contactingthe spinal or joint sample with specific binding partner for A2M, orfragments or complexes thereof, purifying a desired fraction from thesample, and detecting binding between the specific binding partner andmolecules of the sample.

Detection of specific binding of the specific binding partners withmolecules of the sample, when compared to a suitable control, can be anindication that biomarkers are present in the sample. A variety ofmethods to detect specific protein interactions are known in the art andcan be used in the method. Methods include competitive assays andnoncompetitive assays.

Suitable methods include, but are not limited to, Western blot,immunoprecipitation, ELISA and radio-immunoassays. Methods forperforming these and other suitable assays are known in the art. Ingeneral, the specific binding partner used to detect the biomarker willbe delectably labeled, either directly or indirectly. The spinal orjoint sample can be brought into contact with and immobilized on a solidsupport or carrier, such as a membrane (i.e. nitrocellulose) orpolystyrene or magnetic beads that can be capable of immobilizing cells,cell particles, or soluble proteins. The support can then be washed withsuitable buffers, followed by contacting with a detectably-labeledselective binding partner.

In specific binding assays, it can be desirable to minimize the amountof non-specific binding that occurs, particularly when the specificbinding partner can be attached to a substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused. In addition to, or in place of proteinaceous material, variousdetergents and/or salt can be incorporated into the immunoassay tominimize non-specific interactions. Throughout the assays, incubationand/or washing steps can be required after each combination of reagents.Incubation and washing times will depend upon several factors, includingthe assay format, the affinity of the specific binding partner for thebiomarker, the volume of solution, concentrations.

A positive control for an inflammation biomarker can be used in thedetection assays, for example to calibrate the detection assay. A2M canbe from different sources, for example from different species. A2M canbe recombinant, natural or a combination thereof.

In general, protein expression levels can be determined by contacting abiological sample isolated from a subject with binding agents for one ormore of the protein markers; detecting, in the sample, the levels ofpolypeptides that bind to the binding agents; and comparing the levelsof polypeptides in the sample with the levels of polypeptides in acontrol sample. A binding agent can be an entity or composition such asa polypeptide or antibody that specifically binds to a protein marker. Abinding agent can specifically bind to a polypeptide if it reacts and/orinteracts at a detectable level with the polypeptide, but does not reactand/or interact detectably with peptides containing unrelated sequencesor sequences of different polypeptides.

The binding agent can be a ribosome, with or without a peptidecomponent, an RNA molecule, or a polypeptide (e.g., a polypeptide thatcomprises a polypeptide sequence of a protein marker, a peptide variantthereof, or a non-peptide mimetic of such a sequence).

The binding agent can be an antibody specific for a protein marker ofthe invention. Suitable antibodies for use in the methods of the presentinvention include monoclonal and polyclonal antibodies, immunologicallyactive fragments (e.g., Fab or (Fab)₂ fragments), antibody heavy chains,humanized antibodies, antibody light chains, and chimeric antibodies.Antibodies, including monoclonal and polyclonal antibodies, fragmentsand chimeras, can be prepared using methods known in the art (see, forexample, R. G. Mage and E. Lamoyi, in “Monoclonal Antibody ProductionTechniques and Applications”, 1987, Marcel Dekker, Inc.: New York, pp.79-97; G. Kohler and C. Milstein, Nature, 1975, 256: 495-497; D. Kozboret al., J. Immunol. Methods, 1985, 81: 31-42; and R. J. Cote et al.,Proc. Natl. Acad. Sci. 1983, 80: 2026-203; R. A. Lerner, Nature, 1982,299: 593-596; A. C. Nairn et al., Nature, 1982, 299: 734-736; A. J.Czernik et al., Methods Enzymol. 1991, 201: 264-283; A. J. Czernik etal., Neuromethods: Regulatory Protein Modification: Techniques &Protocols, 1997, 30: 219-250; A. J. Czemik et al., Neuroprotocols, 1995,6: 56-61; H. Zhang et al., J. Biol. Chem. 2002, 277: 39379-39387; S. L.Morrison et al., Proc. Natl. Acad. Sci., 1984, 81: 6851-6855; M. S.Neuberger et al., Nature, 1984, 312: 604-608; S. Takeda et al., Nature,1985, 314: 452-454). Antibodies to be used in the methods of theinvention can be purified by methods well known in the art (see, forexample, S. A. Minden, “Monoclonal Antibody Purification”, 1996, IBCBiomedical Library Series: Southbridge, Mass.). For example, antibodiescan be affinity purified by passage over a column to which a proteinmarker or fragment thereof can be bound. The bound antibodies can thenbe eluted from the column using a buffer with a high salt concentration.

Instead of being prepared, antibodies to be used in the methods of thepresent invention can be obtained from scientific or commercial sources.

The binding agent can be directly or indirectly labeled with adetectable moiety. The role of a detectable agent can be to facilitatethe detection step of the diagnostic method by allowing visualization ofthe complex formed by binding of the binding agent to the protein marker(or analog or fragment thereof). The detectable agent can be selectedsuch that it generates a signal which can be measured and whoseintensity can be related or proportional to the amount of protein markerpresent in the sample being analyzed. Methods for labeling biologicalmolecules such as polypeptides and antibodies are well-known in the art(see, for example, “Affinity Techniques. Enzyme Purification B”, Methodsin Enzymol., 1974, Vol. 34, W. B. Jakoby and M. Wilneck (Eds.), AcademicPress: New York, N.Y.; and M. Wilchek and E. A. Bayer, Anal. Biochem.,1988, 171: 1-32).

Specific binding to an antibody, for example, when referring to aprotein or peptide, can be a binding reaction that can be determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies can bind to a particular protein or protein complexat least two times the background and do not substantially bind in asignificant amount to other proteins present in the sample. Specificbinding between a binding agent, e.g., an antibody and a protein, forinstance, a biomarker, can be the ability of a capture- ordetection-agent to preferentially bind to a particular antigen that canbe present in a mixture; e.g., a biological sample. In some embodiments,specific binding can refer to a dissociation constant (KD) that can beless than about 10⁻⁶M; preferably, less than about 10⁻⁵ M; and, mostpreferably, less than about 10⁻⁹ M.

Specific binding assays, including immunoassays, can use a labelingagent to specifically bind to and allow for the detection of the complexformed by the specific binding partner and the detected analyte. A labelor detectable moiety can be a composition detectable by spectroscopic,photochemical, biochemical, radiographic, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The labels can be incorporated into nucleicacids, proteins and antibodies at any position. Any method known in theart for conjugating the antibody to the label can be employed, e.g.,using methods described in Hermanson, Bioconjugate Techniques 1996,Academic Press, Inc., San Diego.

The labeling agent can be a part of the specific binding partner used todetect the analyte. Alternatively, the labeling agent can be a thirdmoiety, such a secondary antibody, which specifically binds to thecomplex formed by the specific binding partner and the detected analyte.Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagent. These proteins exhibit a strong affinity for immunoglobulinconstant regions from a variety of species (see, e.g. Kronval et al., J.Immunol. 111: 1401-1406 (1973); Akerstrom et al., J. Immunol.135:2589-2542 (1985)). The labeling agent can be modified with adetectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well-known to those skilled in the art.

The detectable label can be any material having a detectable physical orchemical property. Many useful detectable labels are known in the artand include any label that can be detectable by spectroscopic,photochemical, biochemical, immunochemical, radiographic, electrical,optical or chemical means. The choice of label can depend on thesensitivity required, the ease of conjugation with the compound,stability requirements, available instrumentation, and disposalprovisions. Useful labels include magnetic beads (e.g., DYNABEADS®),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) can be covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which can be either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize the biomarkers, or secondary antibodies that recognizethe antibodies to the biomarkers.

The molecules can also be conjugated directly to signal generatingcompounds, for example, by conjugation with an enzyme or fluorophore.Enzymes that can be used as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Exemplary fluorescent compounds include, butare not limited to, fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and μMbelliferone. Exemplary chemiluminescentcompounds include, but are not limited to, luciferin and2,3-dihydrophthalazinediones. Means of detecting labels are well knownto those of skill in the art.

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides, fluorescent dyes,chemiluminescent agents, microparticles (such as, for example, quantumdots, nanocrystals, phosphors and the like), enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the binding agents (e.g., antibodies) can beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinylether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents can be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers can be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Protein expression levels in the diagnostic methods of the presentinvention can be determined using immunoassays. Examples of such assaysare radioimmunoassays, enzyme immunoassays (e.g., ELISA),immunofluorescence immunoprecipitation, latex agglutination,hemagglutination, and histochemical tests, which are conventionalmethods well-known in the art. As will be appreciated by one skilled inthe art, the immunoassay can be competitive or noncompetitive. Methodsof detection and quantification of the signal generated by the complexformed by binding of the binding agent with the protein marker willdepend on the nature of the assay and of the detectable moiety (e.g.,fluorescent moiety). Alternatively, the protein expression levels can bedetermined using mass spectrometry based methods or image (including useof labeled ligand) based methods known in the art for the detection ofproteins. Other suitable methods include proteomics-based methods.

Determination of expression levels of nucleic acid molecules in thepractice of the inventive methods can be performed by any suitablemethod, including, but not limited to, Southern analysis, Northernanalysis, polymerase chain reaction (PCR) (see, for example, U.S. Pat.Nos. 4,683,195; 4,683,202, and 6,040,166; “PCR Protocols: A Guide toMethods and Applications”, Innis et al. (Eds.), 1990, Academic Press:New York), reverse transcriptase PCR(RT-PCT), anchored PCR, competitivePCR (see, for example, U.S. Pat. No. 5,747,251), rapid amplification ofcDNA ends (RACE) (see, for example, “Gene Cloning and Analysis: CurrentInnovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, forexample, EP 01 320308), one-sided PCR (Ohara et al., Proc. Natl. Acad.Sci., 1989, 86: 5673-5677), in situ hybridization, Taqman based assays(Holland et al., Proc. Natl. Acad. Sci., 1991, 88:7276-7280),differential display (see, for example, Liang et al., Nucl. Acid. Res.,1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleicacid sequence based amplification (NASBA) and other transcription basedamplification systems (see, for example, U.S. Pat. Nos. 5,409,818 and5,554,527), Qbeta Replicase, Strand Displacement Amplification (SDA),Repair Chain Reaction (RCR), nuclease protection assays,subtraction-based methods, Rapid-Scan™, and the like.

Nucleic acid probes for use in the detection of polynucleotide sequencesin biological samples can be constructed using conventional methodsknown in the art. Suitable probes can be based on nucleic acid sequencesencoding at least about 5 sequential amino acids from regions of nucleicacids encoding a protein marker, and preferably comprise about 15 toabout 50 nucleotides. A nucleic acid probe can be labeled with adetectable moiety, as mentioned above in the case of binding agents. Theassociation between the nucleic acid probe and detectable moiety can becovalent or non-covalent. Detectable moieties can be attached directlyto nucleic acid probes or indirectly through a linker (E. S. Mansfieldet al., Mol. Cell. Probes, 1995, 9: 145-156). Methods for labelingnucleic acid molecules are well known in the art (for a review oflabeling protocols, label detection techniques and recent developmentsin the field, see, for example, L. J. Kricka, Ann Clin. Biochem. 2002,39: 114-129; R. P. van Gijlswijk et al., Expert Rev. Mol. Diagn. 2001,1: 81-91; and S. Joos et al., J. Biotechno 1. 1994, 35:135-153).

Nucleic acid probes can be used in hybridization techniques to detectpolynucleotides encoding the protein markers. The technique cangenerally involve contacting an incubating nucleic acid molecules in abiological sample obtained from a subject with the nucleic acid probesunder conditions such that specific hybridization takes place betweenthe nucleic acid probes and the complementary sequences in the nucleicacid molecules. After incubation, the non-hybridized nucleic acids areremoved, and the presence and amount of nucleic acids that havehybridized to the probes are detected and quantified.

Detection of nucleic acid molecules comprising polynucleotide sequencescoding for a protein marker can involve amplification of specificpolynucleotide sequences using an amplification method such as PCR,followed by analysis of the amplified molecules using techniques knownin the art. Suitable primers can be routinely designed by one skilled inthe art. In order to maximize hybridization under assay conditions,primers and probes employed in the methods of the invention generallyhave at least about 60%, preferably at least about 75% and morepreferably at least about 90% identity to a portion of nucleic acidsencoding a protein marker.

Hybridization and amplification techniques described herein can be usedto assay qualitative and quantitative aspects of expression of nucleicacid molecules comprising polynucleotide sequences coding for theinventive protein markers.

Alternatively, oligonucleotides or longer fragments derived from nucleicacids encoding each protein marker can be used as targets in amicroarray. A number of different array configurations and methods oftheir production are known to those skilled in the art (see, forexample, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974;5,384, 261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554, 501; 5,561,071;5,571,639; 5,593,839; 5,599,695; 5,624, 711; 5,658,734; and 5,700,637).Microarray technology allows for the measurement of the steady-statelevel of large numbers of polynucleotide sequences simultaneously.Microarrays currently in wide use include cDNA arrays andoligonucleotide arrays. Analyses using microarrays are generally basedon measurements of the intensity of the signal received from a labeledprobe used to detect a cDNA sequence from the sample that hybridizes toa nucleic acid probe immobilized at a known location on the microarray(see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and6,271,002). Array-based gene expression methods are known in the art andhave been described in numerous scientific publications as well as inpatents (see, for example, M. Schena et al., Science, 1995, 270:467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93:10614-10619; 1. 1. Chen et al., Genomics, 1998, 51: 313324; U.S. Pat.Nos. 5,143,854; 5,445,934; 5,807,522; 5,837, 832; 6,040,138; 6,045,996;6,284,460; and 6,607,885).

Any biomarker binding agent, such as an antibody, can be labeled with aradiolabel or a fluorescent label. A labeled biomarker binding agent canbe administered into a subject, by any suitable method, such as byinjection. In some embodiments, a labeled biomarker binding agent can beadministered locally, such as to a site of pain or inflammation, forexample, a joint or spine disc. The labeled biomarker binding agent canbe detected by any suitable means known in the art. Exemplaryinstruments that can be used to detect radiolabeled agents orfluorescent agents after administration to a subject include, but arenot limited to, instruments for IVIS Imaging™ (Calipur), bioluminescenceimaging (BLI), fluorescence-lifetime imaging (FLI) microscopy, X-rayradiography, ultrasound imaging, computed tomography (CT) imaging,single-photon emission computed tomography (SPECT), positron emissiontomography (PET), magnetic resonance imaging (MRI), or any combinationthereof. A labeled biomarker agent can bind to its respective biomarkerupon administration of the agent into a subject. In some embodiments,the intensity of the signal from the label and the region in a subject'sbody where the label accumulates can indicate a painful or inflamed discor joint where treatment is needed. For example, a fluorescently labeledantibody to A2M can be injected into a subject and can bind to the A2Mof the subject in a knee joint where the label can accumulate and canindicate the knee joint is in need of treatment.

Therapeutic Methods

Once the site from which the pain can be originating can be identifiedby the presence of A2M, any method known in the art can be used to treatthe pain, or to treat the pathology that can be causing the pain. Forexample, if radiculopathy or discogenic pain or facet pain has beendiagnosed, any number of methods known in the art for treating spinalpain can be applied to treat the patient. Suitable methods include, butare not limited to, laminotomy, laminectomy, discectomy,microdiscectomy, percutaneous discectomy, endoscopic discectomy, laserdiscectomy, foramenotomy, fusion, prolotherapy, other surgicaldecompressions, decompression with fusion with or withoutinstrumentation.

Pain in the spine can also be treated by standard non-surgical methods,including administration of steroidal or non-steroidal anti-inflammatoryagents. Non-steroidal anti-inflammatory (NSAID) agents are well known inthe art. Non-steroidal agents, including NSAIDs such as ibuprofen,aspirin or paracetamol can be used. Steroids, such as glucocorticoids,which reduce inflammation by binding to cortisol receptors, can also beused for treatment.

Any number of methods known in the art for treating joint-related paincan be applied to treat the patient. Suitable methods include surgicaland non-surgical methods including, but not limited to, arthroscopicdebridement or administration of steroidal or non-steroidalanti-inflammatory agents.

Any of the compositions described herein can be used for enhancing thenonspecific inhibition of one or more proteases in a human or non-humananimal experiencing or susceptible to one or more conditions selectedfrom the group of arthritis, inflammation, ligament injury, tendoninjury, bone injury, cartilage degeneration, cartilage injury, anautoimmune disease, back pain, joint pain, joint degeneration, discdegeneration, spine degeneration, bone degeneration, or any combinationthereof. An autologous A2M composition, variant A2M polypeptide, and/oragent that prevents, slows or alters FAC formation can be administeredto an animal to reduce one or more protease activities in an animal.

An autologous A2M composition, variant A2M polypeptide, and/or agentthat prevents, slows or alters FAC formation can be used for inhibitingproteases. An autologous A2M composition and/or a variant A2Mpolypeptide can be used for treatment of pain and inflammationconditions and diseases. An autologous A2M composition and/or a variantA2M polypeptide can be used to prevent, slow, or alter FAC formation. Avariant A2M can be more efficient than a wild-type A2M polypeptide ininhibiting proteases, have a longer half-life, have a slower clearancefactor, or any combination thereof.

Any of the compositions, agents or formulations described herein, suchan autologous A2M composition, variant A2M polypeptide, and/or agentthat prevents, slows or alters FAC formation can be for administrationby parenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, vaginal,rectal, or sublingual), oral, intra-articular or inhalation routes ofadministration. An autologous A2M composition, variant A2M polypeptide,and/or agent that prevents, slows or alters FAC formation can also beadministered using bioerodible inserts, bare-metal stents (BMS), ordrug-eluting stents (DES or coated stents, or medicated stents), and canbe delivered directly to spinal structures, such as intervertebraldiscs, the epidural space and facet joints, or to diarthroidal joints.An autologous A2M composition, variant A2M polypeptide, and/or agentthat prevents, slows or alters FAC formation can be formulated in dosageforms appropriate for each route of administration. An autologous A2Mcomposition, variant A2M polypeptide and/or agent that prevents, slowsor alters FAC formation that are not peptides or polypeptides, canadditionally be formulated for enteral administration.

An autologous A2M composition, variant A2M polypeptide, and/or agentthat prevents, slows or alters FAC formation disclosed herein can beadministered to a subject in a therapeutically effective amount. Theprecise dosage will vary according to a variety of factors such assubject dependent variables, such as age, the injury or pathology beingtreated, and the treatment being affected. The exact dosage can bechosen by the individual physician in view of the patient to be treated.Dosage and administration are adjusted to provide sufficient levels ofthe active moiety or to maintain the desired effect. Additional factorsthat can be taken into account include the severity of the disease, ageof the organism, and weight or size of the organism; diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Short actingpharmaceutical compositions are administered daily whereas long actingpharmaceutical compositions are administered every 2, 3 to 4 days, everyweek, or once every two weeks. Depending on half-life and clearance rateof the particular formulation, the pharmaceutical compositions of theinvention are administered once, twice, three, four, five, six, seven,eight, nine, ten or more times per day.

For some compositions, such as an autologous A2M composition, variantA2M polypeptide, and/or agent that prevents, slows or alters FACformation disclosed herein, as further studies are conducted informationwill emerge regarding appropriate dosage levels for treatment of variousconditions in various subjects, and the ordinary skilled worker,considering the therapeutic context, age, and general health of therecipient, will be able to ascertain proper dosing. The selected dosagedepends upon the route of administration, and on the duration of thetreatment desired. Generally dosage levels can include 0.1 to 40 mg/kgof body weight daily. Generally, for local injection or infusion,dosages can be lower. Depending on the composition and site ofadministration, dosage levels can be between about 1 to 500,000 mg, in avolume between about 0.1 to 10 mL. For example, dosage levels can bebetween about 5 to 450 mg, 5 to 400 mg, 5 to 350 mg, 5 to 300 mg, 5 to250 mg, 5 to 200 mg, 5 to 150 mg, 5 to 100 mg, 5 to 500 mg, 5 to 25 mg,100 to 150 mg, 100 to 200 mg, 100 to 250 mg, 100 to 300 mg, 100 to 350mg, 100 to 400 mg, 100 to 450 mg, or 100 to 500 mg in a volume betweenabout 0.1 to 9 mL, 0.1 to 8 mL, 0.1 to 7 mL, 0.1 to 6 mL, 0.1 to 5 mL,0.1 to 4 mL, 0.1 to 3 mL, 0.1 to 2 mL, 0.1 to 1 mL, 0.1 to 0.9 mL, 0.1to 0.7 mL, 0.1 to 0.6 mL, 0.1 to 0.5 mL, 0.1 to 0.4 mL, 0.1 to 0.3 mL,0.1 to 0.2 mL, 1 to 9 mL, 1 to 8 mL, 1 to 7 mL, 1 to 6 mL, 1 to 5 mL, 1to 4 mL, 1 to 3 mL, or 1 to 2 mL. Normal dosage amounts of variousvariant A2M polypeptides or nucleic acids, or fragment thereof can varyfrom any number between approximately 1 to 500,000 micrograms, up to atotal dose of about 50 grams, depending upon the route ofadministration. Desirable dosages include, for example, 250 ug, 500 ug,1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900mg, 1 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g,2 g, 3 g, 4 g, 5, 6 g, 7 g, 8 g, 9 g, 10 g, 20 g, 30 g, 40 g, and 50 g.

The dose of the variant A2M polypeptide, or fragment thereof, can beadministered to produce a tissue or blood concentration or both fromapproximately any number between 0.1 μM to 500 mM. Desirable dosesproduce a tissue or blood concentration or both of about any numberbetween 1 to 800μM. Preferable doses produce a tissue or bloodconcentration of greater than about any number between 10 μM to about500μM. Preferable doses are, for example, the amount of activeingredient required to achieve a tissue or blood concentration, or both,of 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM,60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM,120 μM, 130 μM, 140 μM, 145 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM,200 μM, 220 μM, 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM,360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500μM.Although doses that produce a tissue concentration of greater than 800μM are not preferred, they can be used with some embodiments of theinvention. A constant infusion of the variant A2M polypeptide, orfragment thereof, can also be provided so as to maintain a stableconcentration in the tissues as measured by blood levels.

Any composition described herein, including an autologous A2Mcomposition, variant A2M polypeptide, and/or agent that prevents, slowsor alters FAC formation can be administered in an aqueous solution byparenteral, intradiscal, intrafacet, intrathecal, epidural or jointinjection. Any composition described herein can be administered directlyinto the area of the spine or joint that can be the source of pain inthe subject. For example, when fibronectin-aggrecan complexes aredetected in the epidural space, a variant A2M polypeptide that inhibitsproteases or that prevents FAC formation can be administered by directinjection into the epidural space. Alternatively, variant A2Mpolypeptide that inhibits proteases or that prevents FAC formation canbe administered by direct injection into the disc space, facet joint, ordiarthroidial joint when fibronectin-aggrecan complexes are detected inthese spaces. In some embodiments, aggrecan can include anynaturally-occurring variants and splice variants of aggrecan, versican,brevican and neurocan, and any variants of aggrecan, versican, brevicanand neurocan due to splicing by different cell types. In someembodiments, fibronectin can include any naturally occurring fibronectinvariants including approximately 20 known splice variants associatedwith a disease or a disorder and fibronectin variants due to differentsplicing by different cell types.

A composition or formulation or agent can also be in the form of asuspension or emulsion. In general, pharmaceutical compositions areprovided including effective amounts of a peptide or polypeptide, andoptionally include pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents sterile water, buffered saline of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; andoptionally, additives such as detergents and solubilizing agents (e.g.,TWEEN® 20, TWEEN® 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. The formulations can belyophilized and redissolved or resuspended immediately before use. Theformulation can be sterilized by, for example, filtration through abacteria retaining filter, by incorporating sterilizing agents into thecompositions, by irradiating the compositions, or by heating thecompositions.

Any composition described herein, including an autologous A2Mcomposition, variant A2M polypeptide, and/or agent that prevents, slowsor alters FAC formation can also be administered in controlled releaseformulations. Controlled release polymeric devices can be made for longterm release systemically following implantation of a polymeric device(rod, cylinder, film, or disc) or injection (microparticles). The matrixcan be in the form of microparticles such as microspheres, wherepeptides are dispersed within a solid polymeric matrix or microcapsules,where the core can be of a different material than the polymeric shell,and the peptide can be dispersed or suspended in the core, which can beliquid or solid in nature. Unless specifically defined herein,microparticles, microspheres, and microcapsules are usedinterchangeably. Alternatively, the polymer can be cast as a thin slabor film, ranging from nanometers to four centimeters, a powder producedby grinding or other standard techniques, or even a gel such as ahydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of any composition described herein, although biodegradablematrices are preferred. These can be natural or synthetic polymers,although synthetic polymers are preferred due to the bettercharacterization of degradation and release profiles. The polymer can beselected based on the period over which release can be desired. In somecases linear release can be most useful, although in others a pulserelease or “bulk release” can provide more effective results. Thepolymer can be in the form of a hydro gel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection which will typically deliver a dosage that canbe much less than the dosage for treatment of an entire body or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

Any of the compositions described herein, such as an autologous A2Mcomposition, variant A2M polypeptide, and/or agent that prevents, slowsor alters FAC formation, can be used in the treatment of a condition ora disease. For example, a condition or disease can be tendon condition,ligament condition, joint injury, spine injury, or inflammation,Alzheimer's disease, cerebral amyloid angiopathy, multiple sclerosis,congenital anti-thrombin deficiency, rheumatoid arthritis, growth ofvarious tumors, coronary or limb ischemia, retinopathies, and regulationof immune response to tumors and viral infections. Others include Acnevulgaris, Alzheimer's disease, arthritis, asthma, acne, allergies andsensitivities, Autoimmune diseases, atherosclerosis, bronchitis, cancer,carditis, Crohn's disease, colitis, chronic pain, cirrhosis, Celiacdisease, Chronic prostatitis, dermatitis diverticulitis, dementia,dermatitis, diabetes, dry eyes, edema, emphysema, eczema, fibromyalgia,gastroenteritis, gingivitis, Glomerulonephritis, Hypersensitivities,hepatitislupus erythematous, acid reflux/heartburn, heart disease,hepatitis, high blood pressure, insulin resistance, Interstitialcystitis, Inflammatory bowel diseases, irritable bowel syndrome (IBS),joint pain/arthritis/rheumatoid arthritis, metabolic syndrome (syndromeX), myositis, nephritis, obesity, osteopenia, osteoporosis, Pelvicinflammatory disease, Parkinson's disease, periodontal disease,polyarteritis, polychondritis, psoriasis, Reperfusion injury, Rheumatoidarthritis, Sarcoidosis, scleroderma, sinusitis, Sjögren's syndrome,spastic colon, systemic candidiasis, tendonitis, Transplant rejection,ulcerative colitcis, UTI's, Vasculitis, and vaginitis.

In some embodiments, an autologous A2M composition, variant A2Mpolypeptide and/or agent that prevents, slows or alters FAC formationthat are not peptides or polypeptides, can be used in the treatment ofcancer. For example, particularly by an autologous A2M composition,variant A2M polypeptide and/or agent that prevents, slows or alters FACformation that are not peptides or polypeptides, can be administereddirectly into a tumor, such as a solid tumor, by injection or anothersuitable means.

An autoimmune disease can be a disease or disorder arising from anddirected against an individual's own tissues or organs or a co-segregateor manifestation thereof or resulting condition therefrom. In many ofthese autoimmune and inflammatory disorders, a number of clinical andlaboratory markers can exist, including, but not limited to,hypergammaglobulinemia, high levels of auto-antibodies, antigen-antibodycomplex deposits in tissues, benefit from corticosteroid orimmunosuppressive treatments, and lymphoid cell aggregates in affectedtissues. Without being limited to any one theory regarding B-cellmediated autoimmune disease, it is believed that B-cells demonstrate apathogenic effect in human autoimmune diseases through a multitude ofmechanistic pathways, including autoantibody production, immune complexformation, dendritic and T-cell activation, cytokine synthesis, directchemokine release, and providing a nidus for ectopic neo-lymphogenesis.

Each of these pathways can participate to different degrees in thepathology of autoimmune diseases. “Autoimmune disease” can be anorgan-specific disease (i.e., the immune response can be specificallydirected against an organ system such as the endocrine system, thehematopoietic system, the skin, the cardiopulmonary system, thegastrointestinal and liver systems, the renal system, the thyroid, theears, the neuromuscular system, the central nervous system, etc.) or asystemic disease that can affect multiple organ systems (for example,SLE, RA, polymyositis, etc.). Preferred such diseases include autoimmunerheumatologic disorders (such as, for example, RA, Sjogren's syndrome,scleroderma, lupus such as SLE and lupus nephritis, polymyositis,dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome,and psoriatic arthritis), autoimmune gastrointestinal and liverdisorders (such as, for example, inflammatory bowel diseases (e.g.,ulcerative colitis and Crohn's disease), autoimmune gastritis andpernicious anemia, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, and celiac disease), vasculitis be (suchas, for example, ANCA-negative vasculitis and ANCA-associatedvasculitis, including Churg-Strauss vasculitis, Wegener'sgranulomatosis, and microscopic polyangiitis), autoimmune neurologicaldisorders (such as, for example, MS, opsoclonus myoclonus syndrome,myasthenia gravis, neuromyelitis optica, Parkinson's disease,Alzheimer's disease, and autoimmune polyneuropathies), renal disorders(such as, for example, glomerulonephritis, Goodpasture's syndrome, andBerger's disease), autoimmune dermatologic disorders (such as, forexample, psoriasis, urticaria, hives, pemphigus vulgaris, bullouspemphigoid, and cutaneous lupus erythematosus), hematologic disorders(such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, posttransfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, ulcerativecolitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome,Graves' disease, IDDM, pernicious anemia, thyroiditis, andglomerulonephritis.

Any of the compositions described herein can be isolated from a bloodsample and can be suitable for delivery into one or more joints or intothe spine. One or more joints can be one or more synovial, diarthrodial,amphiarthrodial, synarthrodial, symphyseal, or cartilaginous joints. Ajoint can be a wrist, spinal, vertebral, cervical, shoulder, elbow,carpal, metacarpal, phalangeal, acromioclavicular, sternoclavicular,scapular, costal, sacroiliac, hip, knee, ankle tarsal, articulations ofa foot or hand, axillary articulations, or a metatarsal.

A joint can refer to any diarthoidal (also called synovial) joints. Ajoint can be any joint containing bone, articular cartilage, a jointcapsule, a synovial tissue lining, or lubricating synovial fluid insidea joint capsule. Cartilage components of a joint can be a chondralcomponent. A component of the knee can be a meniscal component. In someembodiments, a synovial joint can be a shoulder or wrist or ankle or hipor elbow, or the small joints of the fingers or toes. A joint can be anormal joint or a control joint. A normal or control joint can be ajoint that can be an insignificant source of pain to a subject. Thelevel of pain that can be present in a normal joint typically may notimpact the function or quality of the patient's life to the degree thatthe patient seeks medical care. A joint sample or sample from a jointcan be a sample of tissue or fluid from a joint including, but notlimited to, ex vivo and in vivo synovial fluid samples and joint ortissue lavages. A joint sample or sample from a joint can be abiological sample.

Any of the compositions described herein, such as an autologous A2Mcomposition, variant A2M polypeptide, and/or agent that prevents, slowsor alters FAC formation, can be used in the treatment of pain, such aspain associated with a condition or a disease of the current disclosure.

Pain can be radicular pain, radiculopathy, radiculopathic pain andsciatica and can be radiating pain of the extremities which emanatesfrom the spinal root level or “radic” along the path of one or moreirritated lumbar nerve roots. In the case of sciatica, this canoriginate from the L4, L5 and/or L6 or transitional vertebrae if presentand/or sacroiliac spinal nerve roots, which make up the sciatic nerve.Radiating pain can be also possible from the high lumbar discherniations in the l3, l2 or l1 regions or from any cervical nerve rootin the case of a cervical disc herniation, cervical nerve rootirritation or cervical disc degeneration. This pain can differ from painresulting from a facet joint or other spinal structure, which can beclassified as “referred” pain. Radiating pain can be also possible fromthe high lumbar disc herniations in the L3, L2 or L1 regions or cervicalspine regions.

Pain can be discogenic pain and can be spinal related pain thatgenerates from an intervertebral disc. The intervertebral disc suffersfrom reduced functionality in association with a loss of hydration fromthe nucleus pulposus. The reduction in functionality coincides withdamage in the annulus fibrosus. This weakening can lead to anatomiclesions such as bulging, prolapsed, extruded, or sequestered disc. Thisweakening can also lead to possible biochemical lesions resulting fromleakage of the disc contents that can manifest in back pain oraforementioned chemical radiculopathy.

Pain can be facet joint pain or facetogenic pain and can be paingenerating from a facet joint, facet joints, or zygapophysial jointsthat are paired, true synovial joints endowed with cartilage, capsule,meniscoid, and synovial membrane. Spinal-pain or spine related painincludes, but is not limited to, discogenic, facetogenic andradiculopathic pain.

Pain can be acute pain and can be pain lasting up to six months, e.g.,five months, four months, three months, two months, four weeks, threeweeks, two weeks, one week, six days, five days, four days, three days,two days or one day or less. Chronic pain can be pain of duration longerthan six months.

Any subject described herein can be treated with any of the compositionsdescribed herein. In some embodiments, a subject can be diagnosed with acondition or disease before or after being diagnosed with a condition ordisease, such as by the methods described in U.S. Pat. No. 7,709,215 andU.S. Publication No.: US 2010/0098684A1. In some embodiments, a subjectcan be treated with any composition described herein, before or afterbeing diagnosed with a condition or disease.

Subjects

Subjects can include any subject that presents with pain in the spine orjoint. In some embodiments, a subject can be selected for the detectionof A2M. Preferably the subject can be human. Subjects can beexperiencing any pain, such as pain associated with the spine,including, but not limited to, discogenic, facetogenic or radiculopathicpain.

Subjects can be suspected of experiencing pain associated with anyanatomic structure of a joint including, but not limited to, bone,articular cartilage, or the synovial tissue lining. Joints can include,but are not limited to, large diarthrodial (synovial) joints (e.g. knee,hip, shoulder), small diarthrodial (synovial) joints (e.g. elbow, wrist,ankle, zygoapophyseal or facet joints of spine), and amphiarthrodialjoints (e.g. sacroiliac joint, sternoclavicular joint, tempomandibularjoint (“TMJ”)). Subjects can be experiencing acute joint-related pain,or can suffer from chronic joint-related pain. These can be related todegenerative disease (e.g. osteoarthritis), myofascial pain syndromes,inflammatory or crystalline arthritides, or other enthesopathies,tendon/ligament injuries or degeneration, or soft tissue pathologyoutside the musculoskeletal system.

In some embodiments, a subject may have been experiencing joint-relatedor spine-related pain for 30 or 25 weeks or less. In some embodiments, asubject may have been experiencing joint-related or spine-related painfor 20, 15, 10, 8, or 6 weeks, or less. Subjects can be of either sexand can be of any age. Subjects may be experiencing acute or chronicpain.

A subject can be human or non-human animal. For example, the animal canbe a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, horse orgoat. A subject can be a virus, bacterium, mycoplasma, parasite, fungus,or plant, or animal, such as a mammal, for example, a human.

In some embodiments, a subject can be diagnosed as needing treatmentwith any of the compositions described herein. For example, a subjectcan be diagnosed as needing treatment with an A2M enriched sample or anagent that can prevent FAC formation.

Samples

Any of the autologous compositions described herein can be derived froma biological sample. Preferably, the autologous compositions describedherein are isolated from a blood sample and suitable for delivery intoone or more joints or into the spine. Biological samples can alsoinclude sections of tissues such as biopsy samples, frozen sectionstaken for histologic purposes, and lavage samples. A biological samplecan be from a virus, bacterium, mycoplasma, parasite, fungus, or plant.A biological sample can be from an animal, such as a mammal, forexample, a human, non-human primate, rodent, caprine, bovine, ovine,equine, canine, feline, mouse, rat, rabbit, horse or goat.

A biological sample can be a tissue sample or bodily fluid, such as ahuman bodily fluid. For example, the bodily fluid can be blood, sera,plasma, lavage, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, Cowper'sfluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cystfluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretion,mucosal secretion, stool water, pancreatic juice, lavage fluid fromsinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, orμMbilical cord blood. One or more of the biological sample(s) cancomprise a cell, such as a stem cell, undifferentiated cell,differentiated cell, or cell from a diseased subject or subject with aspecific condition. A biological sample can be blood, a cell, apopulation of cells, a quantity of tissue, fluid, or lavasate from ajoint of a subject. A biological sample can comprise cells fromcartilaginous tissue or can be free of cells. A biological sample can besubstantially depleted of a common serum protein, such as, but notlimited to, albumin or IgG. Depletion can comprise filtration,fractionation, or affinity purification.

Biological samples can be collected by any non-invasive means, such as,for example, by drawing blood from a subject, or using fine needleaspiration or needle biopsy. Alternatively, biological samples can becollected by an invasive method, including, for example, surgicalbiopsy.

A biological sample can comprise disease or condition specific proteins.A biological sample can be from a subject with a disease or condition orfrom a subject without a disease or condition. In some embodiments, abiological sample can be from a subject diagnosed with a disease orcondition or from a subject not diagnosed with or without a disease orcondition. A diagnosis can be made by any of the methods describedherein. A biological sample can be from a subject at one time point andanother biological sample can be from a subject at a later or earliertime point, wherein the subject can be the same or a different subject.For example, the subject may have a disease or condition or have beendiagnosed with a disease or condition, and samples can be taken as thedisease or condition progresses. A biological sample can be from asubject pretreatment and another biological sample can be from a subjectat post treatment, wherein the subject can be the same or differentsubject. A biological sample can be from a subject non-responsive totreatment and another biological sample can be from a subject responsiveto a treatment. Biological samples can be from the same or differentspecies. One or more biological samples can be from the same subject orfrom a different subject from which one or more other biological sampleswere obtained.

A spine sample or sample from the spine can be a sample of tissue orfluid from the spine or added to the spine (lavage) including, but notlimited to, spinal disc samples, epidural samples, and facet jointsamples. A spine sample or sample from the spine can be a biologicalsample. Any number of methods known in the art can be used to retrievesample from the spine for the detection of inflammation biomarkers.These methods include, but are not limited to, methods for obtainingsamples from the epidural space, the intervertebral disc space and thefacet joint space. Any number of methods known in the art can be used toobtain joint samples for the detection of inflammation biomarkers.Suitable methods include, but are not limited to, percutaneous or openaspiration, biopsy, or lavage.

The methods of the invention can be applied to the study of any type ofbiological samples allowing one or more biomarkers to be assayed. Abiological sample can be a fresh or frozen sample collected from asubject, or archival samples with known diagnosis, treatment and/oroutcome history.

The inventive methods can be performed on the biological sample itselfwithout or with limited processing of the sample. The inventive methodscan be performed at the single cell level (e.g., isolation of cells fromthe biological sample). Multiple biological samples can be taken fromthe same tissue/body part in order to obtain a representative samplingof the tissue.

Any of the method described herein can be performed on a protein extractprepared from the biological sample. The methods can also be performedon extracts containing one or more of: membrane proteins, nuclearproteins, and cytosolic proteins. Methods of protein extraction are wellknown in the art (see, for example “Protein Methods”, D. M. Bollag etal., 2nd Ed., 1996, Wiley-Liss; “Protein Purification Methods: APractical Approach”, E. L. Harris and S. Angal (Eds.), 1989; “ProteinPurification Techniques: A Practical Approach”, S. Roe, 2nd Ed., 2001,Oxford University Press; “Principles and Reactions o/Protein Extraction,Purification, and Characterization”, H. Ahmed, 2005, CRC Press: BocaRaton, Fla.). Numerous different and versatile kits can be used toextract proteins from bodily fluids and tissues, and are commerciallyavailable from, for example, BioRad Laboratories (Hercules, Calif.), BDBiosciences Clontech (Mountain View, Calif.), Chemicon International,Inc. (Temecula, Calif.), Calbiochem (San Diego, Calif.), PierceBiotechnology (Rockford, Ill.), and Invitrogen Corp. (Carlsbad, Calif.).After the protein extract has been obtained, the protein concentrationof the extract can be standardized to a value being the same as that ofthe control sample in order to allow signals of the protein markers tobe quantitated. Such standardization can be made using photometric orspectrometric methods or gel electrophoresis.

Any of the method described herein can be performed on nucleic acidmolecules extracted from the biological sample. For example, RNA can beextracted from the sample before analysis. Methods of RNA extraction arewell known in the art (see, for example, J. Sambrook et al., “MolecularCloning: A Laboratory Manual”, 1989, 2nd Ed., Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.). Most methods of RNAisolation from bodily fluids or tissues are based on the disruption ofthe tissue in the presence of protein denaturants to quickly andeffectively inactivate RNAses. Isolated total RNA can then be furtherpurified from the protein contaminants and concentrated by selectiveethanol precipitations, phenol/chloroform extractions followed byisopropanol precipitation or cesium chloride, lithium chloride or cesiumtrifluoroacetate gradient centrifugations. Kits are also available toextract RNA (i.e., total RNA or mRNA) from bodily fluids or tissues andare commercially available from, for example, Ambion, Inc. (Austin,Tex.), Amersham Biosciences (Piscataway, N.J.), BD Biosciences Clontech(Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL(Gaithersburg, Md.), and Qiagen, Inc. (Valencia, Calif.).

After extraction, mRNA can be amplified, and transcribed into cDNA,which can then serve as template for multiple rounds of transcription bythe appropriate RNA polymerase. Amplification methods are well known inthe art (see, for example, A. R. Kimmel and S. L. Berger, MethodsEnzymol. 1987, 152: 307-316; J. Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2nd Ed., Cold Spring Harbour Laboratory Press:New York; “Short Protocols in Molecular Biology”, F. M. Ausubel (Ed.),2002, 5th Ed., John Wiley & Sons; U.S. Pat. Nos. 4,683,195; 4,683,202and 4,800,159). Reverse transcription reactions can be carried out usingnon-specific primers, such as an anchored oligo-dT primer, or randomsequence primers, or using a target-specific primer complementary to theRNA for each probe being monitored, or using thermostable DNApolymerases (such as avian myeloblastosis virus reverse transcriptase orMoloney murine leukemia virus reverse transcriptase).

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the art are intended to be within the scope of the invention.Other embodiments are in the claims.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The followingreferences contain embodiments of the methods and compositions that canbe used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007(ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia ofMol. Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Mol. Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Standard procedures of the present disclosure are described, e.g., inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (eds.),Academic Press Inc., San Diego, USA (1987)). Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols inImmunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), and Animal Cell Culture Methods (Methods in CellBiology, Vol. 57, Jennie P. Mather and David Barnes editors, AcademicPress, 1st edition, 1998), which are all incorporated by referenceherein in their entireties.

It should be understood that the following examples should not beconstrued as being limiting to the particular methodology, protocols,and compositions, etc., described herein and, as such, can vary. Thefollowing terms used herein are for the purpose of describing particularembodiments only, and are not intended to limit the scope of theembodiments disclosed herein.

Disclosed herein are molecules, materials, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of methods and compositions disclosedherein. It is understood that when combinations, subsets, interactions,groups, etc. of these materials are disclosed and while specificreference of each various individual and collective combinations andpermutation of these molecules and compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a nucleotide or nucleic acid is disclosed and discussed anda number of modifications that can be made to a number of moleculesincluding the nucleotide or nucleic acid are discussed, each and everycombination and permutation of nucleotide or nucleic acid and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed molecules and compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Those skilled in the art can recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present disclosure which canbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleotide” includes a plurality of such nucleotides; reference to “thenucleotide” is a reference to one or more nucleotides and equivalentsthereof known to those skilled in the art, and so forth.

The term “and/or” shall in the present context be understood to indicatethat either or both of the items connected by it are involved. Whilepreferred embodiments of the present disclosure have been shown anddescribed herein, it can be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions can now occur to those skilled inthe art without departing from the disclosure. It should be understoodthat various alternatives to the embodiments of the disclosure describedherein can be employed in practicing the disclosure. It is intended thatthe following claims define the scope of the disclosure and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Sequences:SEQ ID NO 1: Wild-type A2M precursor protein - complete vector DNA sequence including tagsequences for easier purification. 1CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC 61CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT 121TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA 181CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG 241TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC 301TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG 361ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA 421CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT 481GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG 541TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC 601CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC 661GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC 721CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG 781CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA 841GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC 901TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT 961CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG 1021TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG 1081AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG 1141AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA 1201ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA 1261AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA 1321ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT 1381ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC 1441TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG 1501GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC 1561CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA 1621TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG 1681CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG 1741ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 1801GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA 1861TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG 1921TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT 1981CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG 2041CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT 2101ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG 2161CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG 2221GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG 2281AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC 2341ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC 2401TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC 2461AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC 2521AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT 2581TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC 2641ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG 2701AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT 2761ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC 2821GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT 2881ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT 2941GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG 3001AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG 3061GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA 3121CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG 3181GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG 3241AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC 3301AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG 3361ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC 3421CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC 3481TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG 3541ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC 3601TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG 3661GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT 3721GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC 3781TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC 3841TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT 3901TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT 3961GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG 4021AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA 4081TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT 4141CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCAAGA TGTGCCCACA GCTGCAGCAG 4201TATGAAATGC ACGGACCTGA GGGTCTGCGT GTGGGCTTTT ACGAATCTGA TGTGATGGGA 4261CGTGGTCATG CACGTCTGGT TCATGTCGAG GAACCACACA CCGAAACAGT GCGTAAATAC 4321TTCCCTGAGA CCTGGATTTG GGACCTGGTT GTGGTGAACT CCGCGGGTGT GGCAGAAGTG 4381GGTGTTACCG TCCCGGATAC TATTACCGAA TGGAAAGCAG GTGCCTTCTG TCTGTCTGAG 4441GATGCAGGGC TGGGAATCTC CTCTACAGCC TCTCTGCGCG CGTTTCAGCC CTTTTTCGTC 4501GAACTGACTA TGCCATATAG CGTGATTCGT GGCGAGGCAT TCACTCTGAA AGCTACCGTG 4561CTGAATTACC TGCCCAAGTG CATCCGCGTG AGCGTGCAGC TGGAAGCTAG TCCCGCCTTT 4621CTGGCGGTCC CAGTGGAGAA GGAACAGGCA CCGCACTGCA TTTGTGCTAA CGGCCGGCAG 4681ACTGTTTCCT GGGCCGTCAC CCCCAAATCT CTGGGTAATG TGAACTTCAC CGTTTCAGCA 4741GAGGCTCTGG AAAGCCAGGA GCTGTGCGGC ACCGAAGTCC CATCCGTGCC TGAGCATGGT 4801CGCAAAGATA CAGTCATCAA GCCTCTGCTG GTTGAACCGG AAGGCCTGGA GAAGGAAACT 4861ACCTTTAATT CTCTGCTGTG CCCAAGTGGC GGTGAAGTGT CCGAGGAACT GTCTCTGAAA 4921CTGCCGCCCA ACGTGGTCGA GGAATCTGCC CGTGCGTCAG TTAGCGTCCT GGGGGATATT 4981CTGGGAAGTG CCATGCAGAA TACCCAGAAC CTGCTGCAGA TGCCGTATGG CTGTGGCGAG 5041CAGAATATGG TTCTGTTTGC GCCCAACATC TATGTCCTGG ATTACCTGAA TGAAACACAG 5101CAGCTGACTC CTGAAATCAA AAGCAAGGCA ATCGGGTATC TGAATACCGG ATACCAGCGG 5161CAGCTGAACT ATAAGCACTA CGACGGCTCC TATTCTACCT TCGGCGAACG GTACGGTCGC 5221AATCAGGGGA ACACTTGGCT GACCGCCTTT GTGCTGAAAA CCTTTGCCCA GGCTCGCGCC 5281TATATCTTTA TTGATGAGGC CCATATTACA CAGGCGCTGA TCTGGCTGTC ACAGCGCCAG 5341AAGGACAACG GGTGTTTCCG TAGTTCAGGA AGCCTGCTGA ACAATGCCAT CAAAGGCGGC 5401GTCGAGGATG AAGTGACACT GAGCGCATAC ATTACTATCG CTCTGCTGGA AATCCCTCTG 5461ACAGTGACTC ACCCGGTGGT TCGCAATGCT CTGTTTTGCC TGGAAAGTGC ATGGAAAACA 5521GCTCAGGAAG GCGATCACGG ATCACACGTG TATACTAAGG CACTGCTGGC GTACGCATTC 5581GCTCTGGCCG GCAACCAGGA TAAACGTAAA GAAGTGCTGA AATCACTGAA TGAGGAAGCA 5641GTTAAAAAGG ACAACAGCGT CCACTGGGAA CGGCCGCAGA AACCCAAGGC TCCAGTGGGT 5701CACTTTTATG AGCCTCAGGC ACCGAGTGCT GAGGTGGAAA TGACCTCATA TGTTCTGCTG 5761GCATACCTGA CCGCACAGCC TGCCCCCACA TCAGAAGATC TGACAAGCGC CACTAATATT 5821GTGAAATGGA TCACCAAGCA GCAGAACGCG CAGGGCGGTT TTAGCTCCAC CCAGGACACA 5881GTCGTGGCAC TGCACGCTCT GTCTAAATAT GGGGCAGCTA CCTTCACACG CACTGGAAAG 5941GCCGCGCAAG TGACTATTCA GTCTAGTGGC ACCTTTTCAA GCAAGTTCCA GGTGGATAAC 6001AATAACCGTC TGCTGCTGCA GCAGGTGTCC CTGCCCGAAC TGCCAGGCGA GTACTCTATG 6061AAAGTCACTG GGGAAGGATG CGTGTATCTG CAGACCTCCC TGAAATACAA TATTCTGCCC 6121GAGAAAGAAG AATTTCCATT CGCACTGGGC GTGCAGACCC TGCCTCAGAC ATGCGATGAA 6181CCGAAGGCTC ATACTTCTTT TCAGATCAGT CTGTCAGTGA GCTATACCGG GTCCCGCTCT 6241GCCAGTAACA TGGCGATTGT GGATGTGAAA ATGGTGAGTG GATTCATCCC TCTGAAACCG 6301ACTGTGAAGA TGCTGGAACG GAGTAATCAC GTTTCACGCA CCGAGGTCTC CTCTAACCAT 6361GTGCTGATCT ACCTGGATAA AGTGTCCAAT CAGACACTGT CTCTGTTTTT CACTGTGCTG 6421CAGGATGTCC CCGTGCGTGA CCTGAAACCA GCCATTGTTA AGGTCTATGA TTATTACGAA 6481ACCGACGAGT TCGCGATCGC AGAATACAAC GCGCCGTGCA GCAAAGACCT GGGGAATGCT 6541GACTACAAGG ACGACGACGA CAAGGGGGCA AGCCACCACC ATCACCATCA CTAAGGATCC 6601AAAATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT 6661GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT 6721TGCATCACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG GATTTTGCCG 6781ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC 6841TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA 6901TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG 6961CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA 7021CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC 7081TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA TTCCAGAAGT 7141AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA GCTTGTATAT 7201CCATTTTCGG ATCTGATCAG CACGTGTTGA CAATTAATCA TCGGCATAGT ATATCGGCAT 7261AGTATAATAC GACAAGGTGA GGAACTAAAC CATGGCCAAG CCTTTGTCTC AAGAAGAATC 7321CACCCTCATT GAAAGAGCAA CGGCTACAAT CAACAGCATC CCCATCTCTG AAGACTACAG 7381CGTCGCCAGC GCAGCTCTCT CTAGCGACGG CCGCATCTTC ACTGGTGTCA ATGTATATCA 7441TTTTACTGGG GGACCTTGTG CAGAACTCGT GGTGCTGGGC ACTGCTGCTG CTGCGGCAGC 7501TGGCAACCTG ACTTGTATCG TCGCGATCGG AAATGAGAAC AGGGGCATCT TGAGCCCCTG 7561CGGACGGTGC CGACAGGTGC TTCTCGATCT GCATCCTGGG ATCAAAGCCA TAGTGAAGGA 7621CAGTGATGGA CAGCCGACGG CAGTTGGGAT TCGTGAATTG CTGCCCTCTG GTTATGTGTG 7681GGAGGGCTAA CACGTGCTAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG 7741CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT 7801GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA 7861TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC 7921CAAACTCATC AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC 7981GTAATCATGG TCATTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 8041TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT 8101TACCATCTGG CCCCAGCGCT GCGATGATAC CGCGAGAACC ACGCTCACCG GCTCCGGATT 8161TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 8221CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA 8281ATAGTTTGCG CAACGTTGTT GCCATCGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG 8341GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT 8401TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 8461CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 8521TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC 8581GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA 8641CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC 8701CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 8761TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 8821GAATAAGGGC GACACGGAAA TGTTGAATAC TCATATTCTT CCTTTTTCAA TATTATTGAA 8881GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 8941AACAAATAGG GGTCAGTGTT ACAACCAATT AACCAATTCT GAACATTATC GCGSEQ ID NO 2: Complete vector DNA sequence of the of the acceptor mutant.1 CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC 61CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT 121TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA 181CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG 241TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC 301TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG 361ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA 421CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT 481GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG 541TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC 601CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC 661GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC 721CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG 781CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA 841GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC 901TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT 961CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG 1021TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG 1081AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG 1141AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA 1201ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA 1261AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA 1321ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT 1381ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC 1441TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG 1501GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC 1561CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA 1621TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG 1681CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG 1741ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 1801GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA 1861TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG 1921TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT 1981CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG 2041CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT 2101ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG 2161CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG 2221GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG 2281AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC 2341ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC 2401TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC 2461AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC 2521AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT 2581TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC 2641ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG 2701AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT 2761ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC 2821GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT 2881ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT 2941GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG 3001AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG 3061GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA 3121CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG 3181GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG 3241AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC 3301AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG 3361ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC 3421CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC 3481TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG 3541ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC 3601TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG 3661GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT 3721GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC 3781TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC 3841TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT 3901TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT 3961GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG 4021AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA 4081TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT 4141CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCCAAG ATGTGCCCAC AGCTCGAGCA 4201GTATGAAATG CACGGACCTG AGGGTCTGCG TGTGGGCTTT TACGAATCTG ATGTGATGGG 4261ACGTGGTCAT GCACGTCTGG TTCATGTCGA GGAACCACAC ACCGAAAAGC TTCGTAAATA 4321CTTCCCTGAG ACCTGGATTT GGGACCTGGT TGTGGTGAAC TCCGCGGGTG TGGCAGAAGT 4381GGGTGTTACC GTCCCGGATA CTATTACCGA ATGGAAAGCA GGTGCCTTCT GTCTGTCTGA 4441GGATGCAGGG CTGGGAATCT CCTCTACAGC CTCTCTGCGC GCGTTTCAGC CCTTTTTCGT 4501CGAACTGACT ATGCCATATA GCGTGATTCG TGGCGAGGCA TTCACTCTGA AAGCTACCGT 4561GCTGAATTAC CTGCCCAAGT GCATCCGCGT GAGCGTGCAG CTGGAAGCTA GTCCCGCCTT 4621TCTGGCGGTC CCAGTGGAGA AGGAACAGGC ACCGCACTGC ATTTGTGCTA ACGGCCGGCA 4681GACTGTTTCC TGGGCCGTCA CCCCCAAATC TCTGGGTAAT GTGAACTTCA CCGTTTCAGC 4741AGAGGCTCTG GAAAGCCAGG AGCTGTGCGG CACCGAAGTC CCATCCGTGC CTGAGCATGG 4801TCGCAAAGAT ACAGTCATCA AGCCTCTGCT GGTTGAACCG GAAGGCCTGG AGAAGGAAAC 4861TACCTTTAAT TCTCTGCTGT GCCCAAGTGG CGGTGAAGTG TCCGAGGAAC TGTCTCTGAA 4921ACTGCCGCCC AACGTGGTCG AGGAATCTGC CCGTGCGTCA GTTAGCGTCC TGGGGGATAT 4981TCTGGGAAGT GCCATGCAGA ATACCCAGAA CCTGCTGCAG ATGCCGTATG GCTGTGGCGA 5041GCAGAATATG GTTCTGTTTG CGCCCAACAT CTATGTCCTG GATTACCTGA ATGAAACACA 5101GCAGCTGACT CCTGAAATCA AAAGCAAGGC AATCGGGTAT CTGAATACCG GATACCAGCG 5161GCAGCTGAAC TATAAGCACT ACGACGGCTC CTATTCTACC TTCGGCGAAC GGTACGGTCG 5221CAATCAGGGG AACACTTGGC TGACCGCCTT TGTGCTGAAA ACCTTTGCCC AGGCTCGCGC 5281CTATATCTTT ATTGATGAGG CCCATATTAC ACAGGCGCTG ATCTGGCTGT CACAGCGCCA 5341GAAGGACAAC GGGTGTTTCC GTAGTTCAGG AAGCCTGCTG AACAATGCCA TCAAAGGCGG 5401CGTCGAGGAT GAAGTGACAC TGAGCGCATA CATTACTATC GCTCTGCTGG AAATCCCTCT 5461GACAGTGACT CACCCGGTGG TTCGCAATGC TCTGTTTTGC CTGGAAAGTG CATGGAAAAC 5521AGCTCAGGAA GGCGATCACG GATCACACGT GTATACTAAG GCACTGCTGG CGTACGCATT 5581CGCTCTGGCC GGCAACCAGG ATAAACGTAA AGAAGTGCTG AAATCACTGA ATGAGGAAGC 5641AGTTAAAAAG GACAACAGCG TCCACTGGGA ACGGCCGCAG AAACCCAAGG CTCCAGTGGG 5701TCACTTTTAT GAGCCTCAGG CACCGAGTGC TGAGGTGGAA ATGACCTCAT ATGTTCTGCT 5761GGCATACCTG ACCGCACAGC CTGCCCCCAC ATCAGAAGAT CTGACAAGCG CCACTAATAT 5821TGTGAAATGG ATCACCAAGC AGCAGAACGC GCAGGGCGGT TTTAGCTCCA CCCAGGACAC 5881AGTCGTGGCA CTGCACGCTC TGTCTAAATA TGGGGCAGCT ACCTTCACAC GCACTGGAAA 5941GGCCGCGCAA GTGACTATTC AGTCTAGTGG CACCTTTTCA AGCAAGTTCC AGGTGGATAA 6001CAATAACCGT CTGCTGCTGC AGCAGGTGTC CCTGCCCGAA CTGCCAGGCG AGTACTCTAT 6061GAAAGTCACT GGGGAAGGAT GCGTGTATCT GCAGACCTCC CTGAAATACA ATATTCTGCC 6121CGAGAAAGAA GAATTTCCAT TCGCACTGGG CGTGCAGACC CTGCCTCAGA CATGCGATGA 6181ACCGAAGGCT CATACTTCTT TTCAGATCAG TCTGTCAGTG AGCTATACCG GGTCCCGCTC 6241TGCCAGTAAC ATGGCGATTG TGGATGTGAA AATGGTGAGT GGATTCATCC CTCTGAAACC 6301GACTGTGAAG ATGCTGGAAC GGAGTAATCA CGTTTCACGC ACCGAGGTCT CCTCTAACCA 6361TGTGCTGATC TACCTGGATA AAGTGTCCAA TCAGACACTG TCTCTGTTTT TCACTGTGCT 6421GCAGGATGTC CCCGTGCGTG ACCTGAAACC AGCCATTGTT AAGGTCTATG ATTATTACGA 6481AACCGACGAG TTCGCGATCG CAGAATACAA CGCGCCGTGC AGCAAAGACC TGGGGAATGC 6541TGACTACAAG GACGACGACG ACAAGGGGGC AAGCCACCAC CATCACCATC ACTAAGGATC 6601CAAAATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG 6661TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA 6721TTGCATCACA ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC 6781GATTTCGGCC TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT 6841CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT 6901ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA 6961GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA 7021ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA 7081CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG 7141TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA 7201TCCATTTTCG GATCTGATCA GCACGTGTTG ACAATTAATC ATCGGCATAG TATATCGGCA 7261TAGTATAATA CGACAAGGTG AGGAACTAAA CCATGGCCAA GCCTTTGTCT CAAGAAGAAT 7321CCACCCTCAT TGAAAGAGCA ACGGCTACAA TCAACAGCAT CCCCATCTCT GAAGACTACA 7381GCGTCGCCAG CGCAGCTCTC TCTAGCGACG GCCGCATCTT CACTGGTGTC AATGTATATC 7441ATTTTACTGG GGGACCTTGT GCAGAACTCG TGGTGCTGGG CACTGCTGCT GCTGCGGCAG 7501CTGGCAACCT GACTTGTATC GTCGCGATCG GAAATGAGAA CAGGGGCATC TTGAGCCCCT 7561GCGGACGGTG CCGACAGGTG CTTCTCGATC TGCATCCTGG GATCAAAGCC ATAGTGAAGG 7621ACAGTGATGG ACAGCCGACG GCAGTTGGGA TTCGTGAATT GCTGCCCTCT GGTTATGTGT 7681GGGAGGGCTA ACACGTGCTA CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG 7741GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC 7801TGGAGTTCTT CGCCCACCCC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA 7861ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT 7921CCAAACTCAT CAATGTATCT TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG 7981CGTAATCATG GTCATTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA 8041TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC 8101TTACCATCTG GCCCCAGCGC TGCGATGATA CCGCGAGAAC CACGCTCACC GGCTCCGGAT 8161TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA 8221TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT 8281AATAGTTTGC GCAACGTTGT TGCCATCGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT 8341GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG 8401TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC 8461GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC 8521GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG 8581CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA 8641ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA 8701CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT 8761TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG 8821GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATATTCT TCCTTTTTCA ATATTATTGA 8881AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT 8941AAACAAATAG GGGTCAGTGT TACAACCAAT TAACCAATTC TGAACATTAT CGCGSEQ ID NO 3: Amino Acid Sequence of Tagged wild-type human A2M 1MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV 61SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV 121MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS 181FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT 241ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF 301YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR 361QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT 421VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY 481ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL 541LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAELRVTAA PQSVCALRAV 601DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS 661TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLQQYEMHGP EGLRVGFYES DVMGRGHARL 721VHVEEPHTET VRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI 781SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE 841KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI 901KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ 961NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH 1021YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF 1081RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH 1141GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ 1201APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA 1261LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG 1321CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI 1381VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR 1441DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHHHSEQ ID NO 4: Amino Acid Sequence of the Acceptor Mutant. 1MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV 61SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV 121MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS 181FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT 241ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF 301YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR 361QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT 421VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY 481ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL 541LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV 601DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS 661TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLEQYEMHGP EGLRVGFYES DVMGRGHARL 721VHVEEPHTEK LRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI 781SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE 841KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI 901KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ 961NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH 1021YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF 1081RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH 1141GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ 1201APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA 1261LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG 1321CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI 1381VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR 1441DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHH HSEQ ID NOs 5-66: Amino Acid Sequences of Variant Bait Regions.SEQ ID NO 5:  LEQYEMHGPE GLRVGKEEEG LGSIPENFFG VSELEGRGSK LSEQ ID NO 6:  LEQYEMHGPE GLRVGIPENF FGVSELEGRG SKEEEGLGSK LSEQ ID NO 7:  LEQYEMHGPE GLRVGSELEG RGSKEEEGLG SIPENFFGVK LSEQ ID NO 8:  LEQYEMHGPE GLRVGKEEEG LGSSELEGRG STAQEAGEGK LSEQ ID NO 9:  LEQYEMHGPE GLRVGIPENF FGVFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 10: LEQYEMHGPE GLRVGKEEEG LGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 11: LEQYEMHGPE GLRVGSELEG RGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 12: LEQYEMHGPE GLRVGEAIPM SIPFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 13: LEQYEMHGPE GLRVGTAQEA GEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 14: LEQYEMHGPE GLRVGVSQEL GQRFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 15: LEQYEMHGPE GLRVGTEGEA RGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 16: LEQYEMHGPE GLRVGTSEDL VVQFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 17: LEQYEMHGPE GLRVGEGEGE GEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 18: LEQYEMHGPE GLRVGGEEGV EEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 19: LEQYEMHGPE GLRVGGARGL EGFYESDVMG RGHARLVHVE EPHTKLSEQ ID NO 20: LEQYEMHGPE GLRVGGPPGL APGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 21: LEQYEMHGPE GLRVGGEPEG AKGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 22: LEQYEMHGPE GLRVGEEEGG GFYESDVMGR GHARLVHVEE PHTKLSEQ ID NO 23: LEQYEMHGPE GLRVGGYPGS SRGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 24: LEQYEMHGPE GLRVGGARGL EGGFAGLPNG GEEGVEEGKLSEQ ID NO 25: LEQYEMHGPE GLRVGESESE GGGGGSLLGE FEVEGGFAGL PNGKLSEQ ID NO 26: LEQYEMHGPE GLRVGGFKEG VEGEIEEGGG FKEGVEGKLSEQ ID NO 27: LEQYEMHGPE GLRVGESESE GGFAGLPNGK EEEGLGSIPE NFFGVKLSEQ ID NO 28: LEQYEMHGPE GLRVGIPENF FGVTSEDLVV QEAIPMSIPK LSEQ ID NO 29: LEQYEMHGPE GLRVGEAIPM SIPTSEDLVV QIPENFFGVK LSEQ ID NO 30: LEPAGAARGE SESEGGFFGF PIGERESTGG DRGLPIGENE AGGKLSEQ ID NO 31: LETEGRGERE AQGEFPEVEG EEEGGGPEKE TGGEREAQGK LSEQ ID NO 32: LEARGLEGGG GGSLLGGYPG SSRGGFKEGV EGGPAGAARG KLSEQ ID NO 33: LEPGLAPGGE EGVEEGGPEE GVEEGGFKEG VEGEPESSGK LSEQ ID NO 34: LEEGEARGST AQEAGEGPKE EEGLGSSELE GRGSPVSQEL GQRKLSEQ ID NO 35: LEAQEAGEGK EEEGLGSPVS QELGQRSELE GRGSPTEGEA RGSKLSEQ ID NO 36: LEEEEGLGSK EEEGLGSPKE EEGLGSKEEE GLGSPKEEEG LGSKLSEQ ID NO 37: LEELEGRGSK EEEGLGSIPE NFFGVFYESD VMGRGHARLV HVEEPHTKLSEQ ID NO 38: LEENFFGVTE GEARGSPTSE DLVVQKEEEG LGSEAIPMSI PKLSEQ ID NO 39: LEIPMSIPKE EEGLGSIPEN FFGVTEGEAR GSPTSEDLVV QKLSEQ ID NO 40: LELQQYEMHG PEGLRVGEAI PMSIPIPENF FGVKEEEGLG SKLSEQ ID NO 41: LEEEGVEEGK EEEGLGSGPA GAARGSELEG RGSPTEGEAR GSKLSEQ ID NO 42: LEPESSGEAI PMSIPTSEDL VVQIPENFFG VEAEGTGGER GVLGKLSEQ ID NO 43: LEGGGSLLGE PEPEGEREAQ GGVEGVELGG FKEGVEGEQE GRGKLSEQ ID NO 44: LESQELGQRE SESEGSELEG RGSGFKEGVE GKEEEGLGSG FFGFPIGKLSEQ ID NO 45: LEQYEMHGPK EEEGLGSSEL EGRGSEAIPM SIPTIPENFF GVVEEPHTKLSEQ ID NO 46: LEQYEMHGPS ELEGRGSIPE NFFGVEAIPM SIPTSEDLVV QIVEEPHTKLSEQ ID NO 47: LEQYEMHGPE GEGEGEGIPE NFFGVSEDLV VQISELEGRG SVEEPHTKLSEQ ID NO 50: LEQYEMHGPI PENFFGVSEL EGRGSEAIPM SIPTEGEGEG EGVEEPHTKLSEQ ID NO 51: LEQYEMHGPS ELEGRGSEAI PMSIPTKEEE GLGSIPENFF GVVEEPHTKLSEQ ID NO 52: LEQYEMHGPE AIPMSIPTEG EGEGEGIPEN FFGVSEDLVV QIVEEPHTKLSEQ ID NO 53: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTVEEPHTKLSEQ ID NO 54: LEQYEMHGPE GEGEGEGISE DLVVQIPENF FGVKEEEGLG SVEEPHTKLSEQ ID NO 55: LEQYEMHGPE GEGEGEGIPE NFFGVSELEG RGSSEDLVVQ IVEEPHTKLSEQ ID NO 56: LEQYEMHGPI PENFFGVEGE GEGESELEGR GSSEDLVVQI VEEPHTKLSEQ ID NO 57: LEQYEMHGPS ELEGRGSIPE NFFGVKEEEG LGSSEDLVVQ IVEEPHTKLSEQ ID NO 58: LEQYEMHGPI PENFFGVSEL EGRGSSEDLV VQIKEEEGLG SVEEPHTKLSEQ ID NO 59: LEQYEMHGPK EEEGLGSIPE NFFGVSELEG RGSEGEGEGE GVEEPHTKLSEQ ID NO 60: LEQYEMHGPS EDLVVQIKEE EGLGSIPENF FGVSELEGRG SVEEPHTKLSEQ ID NO 61: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVKEEEGLG SVEEPHTKLSEQ ID NO 62: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTEPHTKLSEQ ID NO 63: LEQYEMHGPE GEGEGEGIPE NFFGVEAIPM SIPTSELEGR GSEPHTKLSEQ ID NO 64: LEQYEMHGPE AIPMSIPTSE LEGRGSIPEN FFGVEGEGEG EGEPHTKLSEQ ID NO 65: LEQYEMHGPS ELEGRGSIPE NFFGVEGEGE GEGKEEEGLG SVEEPHTKLSEQ ID NO 66: LEQYEMHGPI PENFFGVSED LVVQIEGEGE GEGEAIPMSI PTEPHTKL

EXAMPLES Example 1—Generation and Selection of HEK293 Clones ExpressingRecombinant A2M

Recombinant A2M wild type sequence was expressed in HEK293F cells.Hek293F cells are plated adherently and allowed to attach overnight.Cells are transfected with XTreme Gene HP (Roche) and DNA in a 6 uLreagent: 2 ug DNA ratio. Cells are grown for 48 hours at 5% CO2 and 37degrees Celsius. Forty-eight hours after transfection media samples aretaken to confirm success of the transfection via an ELISA assay thatquantifies A2M protein. Cells are split so as to be in logarithmicgrowth phase and selection antibiotic (blasticidin) is added at 10 μg/mL(selection concentration determined experimentally). Cells are selectedin antibiotic until all of the negative control cells are dead (usuallyabout 4 to 5 days). Another media sample is taken at this point toconfirm that this newly established pool is still producing protein.Upon confirmation of protein production cells are plated at a density of−100 cells/10 cm dish with 7.5 μg/mL blasticidin (maintenanceconcentration determined experimentally). This plating density is sparseenough that cells will be spaced far enough apart to allow each cell togrow into an individual colony. These colonies are collected usingcloning cylinders (Sigma) and plated in a 24 well plate to allow furthercell growth. Once cells become confluent in the 24 well plate an ELISAis performed on a media sample again to screen for the highest producingclone. High-expressing clones were selected and used for production ofA2M. The chosen clones were expanded and adapted to suspension (FIG. 3).Suspension adaption was completed by slowly changing the media to aserum-free media while the cells are in shaker flasks. Once the cultureis in suspension, protein can be collected by simply spinning the cellsout of the media. The A2M containing supernatants were subjected topurification for A2M. The higher cell number per volume of media resultsin a higher protein concentration per milliliter of media. High puritysamples were obtained after two chromatography methods. A yield of ˜12mg/L (adherent pool) was typical (FIG. 15).

Example 2—Inhibition of ADAMTS-5- and ADAMTS-4-Induced Damage ofCartilage with A2M

Bovine Cartilage Explants (BCEs) were treated with 500 ng/ml ADAMTS-5 orADAMTS-4 for 2 days, with a 3-fold serial dilution of purified A2M (FIG.7A, B). Concentration of A2M tested were 100, 33.3, 11.1, 3.7, 1.2, 0.4μg/mL. The A2M inhibited cartilage catabolism in a concentrationdependent manner. The IC50 for inhibiting 500 ng/ml of ADAMTS-5 wascalculated to be ˜7 μg/ml A2M (a 1:1 molar ratio). Maximum inhibitionwas observed in ˜90% with 100 μg/ml A2M (a 14:1 molar ratio). The A2Mwas shown to block formation of Aggrecan G3 fragments (FIG. 7A, B) andFAC formation (FIG. 9).

Example 3—Comparison of APIC Retentate and Filtrate

Fresh cartilage was treated with APIC containing ˜7 mg/ml A2M. Cartilagecatabolism was efficiently blocked by 1% v/v of the Retentate of theAPIC production process (concentration of proteins >500 kDa in size),but not by the Filtrate (contains proteins <500 kDa), even at 5% v/v(FIG. 10). The chondroprotective effects of APIC were dose dependent.The inability of Filtrate to protect cartilage from catabolism byADAMTS-5 demonstrates that APIC concentrates >99% of the protectivefactors of autologous blood.

Example 4—APIC Blocks Cartilage Catabolism in an Osteoarthritis Model

Fresh cartilage was treated with TNF-α or IL-1βeta to inducechondrocytes to secrete proteases, similar to the pathology ofosteoarthritis (FIG. 6). Cartilage catabolism was detected as increasedsulfated glycosaminoglycans (sGAG) in the culture media. Treatment withpro-inflammatory cytokines induced cartilage catabolism which treatmentwith APIC was shown to block in a dose-dependent manner.

Example 5—Cytokine Profile of Monocytes Treated with APIC

THP-1 monocyte cells were treated with or without APIC for 2 days andthe activation of the cells was monitored by secretion of cytokines andgrowth factors into the medium. THP-1 did not show a change in thecytokines profile tested (FIG. 11). Similar results were seen in E6-1T-cells and SW982 fibroblast cells.

Example 6—Design and Synthesis of Tagged Wild-Type A2M ExpressionConstruct

A DNA sequence coding for the wild-type A2M precursor protein (SEQ IDNO. 1) was synthesized by GenScript based on the RefSeq amino acidsequence of human A2M precursor protein (RefSeq #NP_000005.2) (SEQ IDNO. 3). The codons used in the construct were optimized by GenScript formammalian codon usage bias, GC content, CpG dinucleotide content, mRNAsecondary structure, cryptic splicing sites, premature polyadenylationsites, internal chi and ribosome binding sites, negative CpG islands,RNA instability motifs, repeat sequences, and restriction endonucleasesites. A sequence encoding a fusion tag (DYKDDDDKGASHHHHHH) was added tothe natural end of the protein sequence, followed by a STOP codon. Theexpression construct was given a Kpn1 restriction site at the 5′ end anda BamH1 restriction site at the 3′ end. This construct was cloned into apUC57 vector. The insert encoding the expression construct was extractedfrom the pUC57 vector via double digestion with Kpn1 and BamH1 followedby agarose gel electrophoresis and gel extraction of the fragment. Thisinsert was ligated into a pJ608 mammalian expression vector (DNA 2.0)behind a cytomegalovirus (CMV) promoter (FIG. 23) and transformed intoE. coli strain GC10 (Genessee Scientific). This step is performed tomaintain and propagate the vector. The sequence of the expressionconstruct was verified by DNA sequencing (Genewiz).

Example 7—Design of Acceptor Construct for Variable Bait Regions

The wild-type expression construct was mutated to allow switching ofbait region sequences by first introducing Xho1 and HindIII restrictionsites flanking the sequence encoding the bait region. This was done viatwo sequential site-directed mutagenesis reactions using the wild-typeexpression construct as the template. The sequence of the mutant“acceptor” construct was verified by DNA sequencing of the bait regionby Genewiz (SEQ ID NO 2). The corresponding amino acid sequence is SEQID No 4. The mutations in the DNA sequence necessarily result in threeamino acid substitutions in the protein Q693E on the N-terminal side ofthe bait region and T730K and V731L on the C-terminal of the baitregion. These mutations could not be avoided because the natural DNAsequence does not have restriction endonuclease sites that could be usedto remove the bait sequence. These mutations are included in the newbait regions design. The preservation of function of the acceptor mutantwas verified by its ability to inhibit trypsin (see below), and it wastested versus other proteases as part of the evaluation of the designedbait regions.

Example 8—Design and Creation of Variable Bait Region ExpressionConstructs

60 novel bait region amino acid sequences (SEQ ID NOs: 5-66) weredesigned based on the known cleavage sites of human aggrecan byADAMTS-4, ADAMTS-5, and various MMPs (Fosang et al., Eur. Cells andMat., Vol. 15, 2008, pp. 11-26) (Table 1). Some constructs retained partor the entirety of the wild-type A2M bait sequence, but with aninsertion of non-native amino acids (SEQ ID NOs: 5-66). Several pUC57plasmids, each containing DNA insert sequences encoding between one andsix bait region sequences, were synthesized by GenScript and deliveredto us as a lyophilized powder. Each insert sequence contains an Xho1site at the 5′ end and a HindIII site at the 3′ end for ligation intothe acceptor construct. Each insert plasmid, along with the acceptorplasmid, was reconstituted in water and double digested overnight with20 U of Xho1 and HindIII to liberate the insert sequences, and thedigested plasmids were separated by electrophoresis on a 1% agarose geland visualized under UV light. Bands corresponding to the insert andacceptor length were extracted from the gel via a Qiagen Qiaquick GelExtraction Kit as per the kit instructions. The concentration of DNAobtained from each extraction was determined using a Qubit fluorimeter(Invitrogen). Ligation of inserts into the region of the acceptorencoding the bait region was undertaken in a semi-random fashion, bymixing the extracted insert fragment(s) from each insert vectordigestion with 50 ng of digested acceptor plasmid in a 3:1 molar ratioof insert:plasmid. Ligation was achieved using a Quick Ligation kit (NewEngland Biolabs) according to the kit instructions. The mixture ofligated plasmids was then transformed into E. coli strain GC10 (GenesseeScientific) and spread onto Luria broth/agar plates containing 100 μg/mLampicillin to generate single colonies of transformants. 5 mL Luriabroth cultures of individual colonies from each ligation reaction weregrown and the plasmid DNA contained within each extracted via a QiagenQiaPrep miniprep kit according to the kit instructions. These plasmidswere sent to Genewiz for sequence confirmation using a primer thatanneals to the sequence of the A2M construct just upstream of the baitregion. The individual chromatogram traces were analyzed for thepresence of heterogeneity in the sequence, and the sequences of theindividual inserts confirmed.

Example 9—Expression of A2M Variants

A2M variants were expressed in HEK293F cells (Gibco) by transienttransfection of each construct in suspension cells. Cells were grown toa density of 550,000 cells/mL in a Erlenmeyer cell culture flaskcontaining 20 mL of FreeStyle F17 medium (Invitrogen) containing 1×GlutaMax (Gibco) on a rotator at a speed of 125 rpm inside a 37° C.incubator containing an 8% CO2/air mixture. Cells were transfected bymixing 20 μg of plasmid DNA of each construct (wild-type or variant) ina 1:2 (w/v) ratio with TransIT Pro plus 10 μL TransIT Boost (Mirus) 15minutes before addition to media. Cells were maintained in the sameconditions for three days after transfection before the media containingsecreted recombinant protein was removed for protein purification (FIG.3).

Example 10—Purification of A2M Variants

Since the A2M expression construct encodes the precursor A2M protein,the expressed and processed recombinant protein is secreted into thecell culture medium via the natural A2M secretion signal. Secretedrecombinant wild-type A2M and A2M bait region variants were purifiedfrom the transfected cell culture media by Immobilized Metal AffinityChromatography using the 6×His tag at the C-terminus of each construct.The media removed from the transfected cells was centrifuged at 17,500Gfor 15 minutes to remove all cells. Imidazole was added to the clarifiedmedia to a final concentration of 10 mM. 1 mL of HisPur Cobalt resinslurry (Pierce) was added to the sample and allowed to equilibrate withshaking on a rocker at 4° C. for one hour. The beads were collected bycentrifugation at 700G for 2 minutes and the supernatant discarded. Thebeads were washed three times in 10 mL of a buffer of 50 mM Tris-Cl, 150mM NaCl, 10 mM imidazole, pH 7.4, each time the beads were collected bycentrifugation at 700G, and the supernatant removed and discarded. Theprotein was eluted by mixing of 2 mL of elution buffer (wash buffercontaining 200 mM imidazole) with the beads and centrifuging for 2minutes at 700G. The supernatant was collected and retained, and theelution repeated a total of three times. The purified proteins containedin the sample were then concentrated to 100 μL volume (typically between100 μg/mL and 600 μg/mL—) using an Amicon spin filter with a NMCO of 100KDa. During concentration the imidazole containing buffer wasexhaustively exchanged for 50 mM HEPES, 150 mM NaCl, 10 mM CaCl₂, 100 μmZnCl₂, 0.05% (w/v) Brij-35, pH 7.4 (HNZCB buffer). The concentration ofthe protein was determined using BCA (Pierce) and 660 nm (Pierce)assays. 1 μg of each purified protein was mixed with reducing SDS-PAGEloading buffer, heated for five minutes at 95° C., and loaded onto a7.5% Tris-glycine SDS-PAGE stain-free gel (Bio-rad). The gel wasdeveloped by exposing to UV light for five minutes, and a picture takenof the total protein bands. The purity of the recombinant A2M wasestimated to be consistently greater than 90% across all variants andwild-type proteins (FIG. 15).

Example 11—Screening of Inhibition Abilities of ADAMTSs and MMPs by A2MVariants

Forty A2M variants containing SEQ ID Nos.: 5-44 and the tagged wild-typeprotein were screened for their comparative ability to inhibitproteolysis of a recombinant IDG fragment of human aggrecan whichconsist of the G1, G2, and interglobular domains (R&D) by ADAMTS-4,ADAMTS-5, and MMP13. Screening the effectiveness of these variants forthe inhibition of each of these enzymes was done in the same mannertaking in consideration the rate of the proteolytic activity of eachprotease. The amount of IGD fragment in each sample was held constant at0.1 μg, whereas the amount of protease varied depending on the activityof the protease toward IGD fragment. Since each of the variants andwild-type A2M vary greatly in the kinetics of bind to each protease,some showed complete inhibition with no pre-incubation of A2M with theprotease, where others showed some inhibition if incubated with theprotease for 10 minutes, and others showed no inhibition even after apre-incubation of A2M with the protease. Two independent assays wereperformed on each A2M variant: one in which the protease, IGD fragment,and A2M were all added at the same time (no pre-incubation), and one inwhich the protease and A2M were pre-incubated at room temperature forten minutes before addition of the IGD fragment, in order to detectslower inhibitors binding to the proteases. For the experiment with nopre-incubation of protease with A2M, 5 μL of 150 nM tagged wild-type A2Mor an A2M variant in HNZCB buffer was added to a microcentrifuge tube. 5μL of 40 μg/mL IGD fragment was then added to the same tube and mixed.Finally 5 μL of 150 nM (ADAMTS-4 and ADAMTS-5, a 1:1 A2M:protease molarratio) or 75 nM (MMP13—a 2:1 A2M:protease molar ratio) protease wasadded to the tube. For the experiment with a 10 minute pre-incubation, 5μL of each A2M was mixed with 5 μL of protease 10 minutes beforeaddition of 5 μL of IGD fragment. All samples were incubated at 37° C.for one hour before being stopped by addition of 2× reducing SDS-PAGEloading buffer (Bio-rad) and heating for 5 min. at 95° C. 15 μL of eachsample was loaded onto a 7.5% Tris-Glycine Stain Free Gel (Bio-Rad) andrun at 150 V for 1 hour. Total protein was visualized and imaged underUV light as per gel instructions. The proteins were then blotted onto anitrocellulose membrane via an iBlot (Invitrogen) dry blotting systemusing a transfer time of seven minutes, blocked for one hour using TBScasein blocking solution (Bio-rad), and probed using an anti-IGDfragment goat polyclonal antibody (R&D Biosystems catalog # AF1220) at aconcentration of 0.1 μg/mL in TBS-T. The blot was washed three timeswith TBS-T and probed with an HRP-conjugated anti-goat IgG polyclonalantibody (Sigma catalog #A5420) at 0.1 μg/mL in casein blockingsolution. The blots were developed using ECL Plus chemiluminescence kits(Pierce) according to the manufacturer instructions. The Western blotswere imaged in a ChemiDoc imager system (Bio-rad). Each IGD fragmentband on the Western (intact and degraded IGD fragment) was quantifiedusing ImageLab software. The amount of degradation of IGD fragment inthe presence of each A2M variant was quantified by comparing theintensities of the degraded and intact IGD fragment bands (FIGS. 16-20),and the inhibitory capacity of each variant was compared to a wild-typeA2M sample that was prepared along with each batch of variants. Fromthis initial round of screening, eight variants were selected forfurther screening against MMP1, MMP2, MMP3, MMP8, MMP9, MMP12, andCathepsin K (all enzymes are recombinant human constructs and purchasedfrom R&D). The comparison of the inhibitory capacity of each variant wasdone by taking the ratio of the intensity of the degraded band to theintact band with the exception of MMP9 and MMP13 which degraded IGDfragment in such a manner that cleaved fragments did not appear on theWestern blot. In these cases the comparison was done based solely on theintensity of the remaining intact IGD fragment band. Additionally,ADAMTS-1 and MMP7 only cleaved the IGD fragment perceptibly; therefore,accurate inhibition measurements could not be quantified. In these casesall of the variants were judged to be essentially equivalent towild-type with respect to these two proteases. After evaluating allinhibition data, four variants were selected based on improved or atleast equivalent inhibition characteristics against all proteases tested(FIGS. 17-21) or a mixture of proteases known to degrade cartilage (FIG.22).

Example 12—Screening of A2M Variants Vs. Trypsin and Chymotrypsin

To verify that the four selected A2M variants are still capable ofinhibiting the general proteases trypsin and chymotrypsin to a similardegree as the wild-type protein, the variants were tested in afluorescent proteolysis assay (Twining, S. S., Anal. Biochem. Vol. 143,1984, pp. 30-34). In this assay, one monitors the increase influorescence emission from a FITC-labeled protein substrate that iscaused by a proteolysis-dependent release of the fluorophore. Twoexperiments were done on each variant: one in which the molar ratio ofA2M:protease is held at 1:1, and another in which the A2M is reduced to0.5:1. 40 μL of wild-type or variant A2M at a concentration of 100 nM(for the 1:1 ratio) or 50 nM (for the 0.5:1 ratio) in HNZCB buffer wasmixed with 100 μL of bovine trypsin (Sigma) at 40 nM and incubated atroom temp for 5 minutes. Into this mixture 70 μL of 40 μg/mL FTC-caseinsubstrate (Pierce) was added, mixed, and immediately pipetted into threewells of a 384 well plate (65 μL/well) The plate was placed into a CaryEclipse fluorimeter and read in kinetic mode (single wavelength) withexcitation wavelength of 485 nm and emission wavelengths of 519 nm forfifteen minutes, during which time the rate of casein degradation by theprotease remains approximately linear. The emission intensity wasaveraged for the three sample wells, plotted vs. time, and a straightline fitted to the data from each sample and control (FIG. 18, left).The slope of the fitted line was taken as a measure of the proteaseactivity remaining in solution. Comparison of the four chosen A2Mvariants to the wild-type protein shows that the variants are allcapable of inhibiting trypsin and chymotrypsin approximately equally tothe wild-type A2M (FIG. 18, right).

Example 13—Preparation of Blood for Autologous Therapy

120 mL of whole human blood was obtained from a subject by venipuncture.38 mL aliquots of the blood were collected into two or more hematologiccollection bottles with a suitable volume of citrate dextrose solution A(“ACD-A”) in each collection bottle. The collection bottles withblood/ACD-A were placed into a fixed angle rotor centrifuge, andcentrifuged at predetermined velocities and times under ambienttemperature conditions. Approximately 15 mL of plasma was aliquoted fromeach tube with a serological pipette, leaving approximately 1 mL, ofplasma above the level of the buffy coat so as not to disturb theprecipitated cells. This process was repeated for the collection bottlesin one or more centrifuge spin cycles to yield a volume 45 mL of totalplasma from a total blood draw of 120 mL. The plasma was pooled into aseparate sterile hematologic collection bag. The compositions describedherein can be mixed with autograft or allograft tissue, such as bone,before administration to a subject.

Example 14—Pump Drive for Systems

This example describes the operating characteristics and limits for aPump Drive (Pump Drive, Fixed Speed, 100-240 VAC) for use with theEasy-Load II Pump Head. This Pump Drive is intended to process APIC PRPthrough the filtration procedure. The Pump Drive is not patientconnected. The APIC PRP will be removed after the filtration process iscomplete and collected through the use of a syringe. The following arefeatures along with an indication of how the pump drive will meet theserequirements.

FEATURE: 100-240 VAC, 50/60 Hz operations. IMPLEMENTATION: A medicalgrade, auto-sensing, switching power supply will be incorporated toallow 100-240 VAC operations

FEATURE: IEC Power Cord Connector. IMPLEMENTATION: The Pump Drive willbe supplied with a 10′ long hospital grade power cord. The power cordwill connector to the Pump Drive via an IEC input connector on themedical grade AC inlet.

FEATURE: Power Indicator, Complete light & 2-Line, 15 character digitaldisplay. IMPLEMENTATION: No separate power indicator or “complete” lightwill be implemented. A 2-Line LCD display will be used to display PumpDrive operations. When the display is illuminated, power is ON. When anoperational cycle is “complete”—a message will be displayed on the2-Line LCD display.

FEATURE: Power (On/Off) switch on front of unit. IMPLEMENTATION: Power(On/Off) switch will be located on the rear of the Pump Drive for safetyand EMC purposes. The rear location will help minimize and ensure allcreepage and clearance distances are met with regard to the AC mains.

FEATURE: Interlock (motor disable when pump head open). IMPLEMENTATION:An interlock will be incorporated into the pump head and pump drive todisable the motor when the occlusion bed is opened and the rotorassembly is exposed during tube set changes.

FEATURE: 3.1 ml/sec±2%. IMPLEMENTATION: A digital motor control systemwith encoder feedback will be utilized to ensure the motor speed isregulated to within ±2%.

FEATURE: Masterflex L/S Easy-Load II Pump Head: Single channel, fixedocclusion; 4-roller, stainless steel rotor; Compatible with tubing size16, ⅛″ ID; Material: Polyphenylene Sulfide; Automated tubing retainers.IMPLEMENTATION: An Easy-Load II with a standard thin wall, stainlesssteel rotor assembly will be used. The Easy-Load II pump headaccommodates all of the requested features.

FEATURE: Control Panel with Stop, Start/Confirm buttons and Up/Downarrows.

IMPLEMENTATION: A—5 key Keypad will be utilized for operator entries.Note—to reduce lead time, an existing 7-key keypad will be utilized onthe prototype Pumps.

FEATURE: Operations—Count Down timer. IMPLEMENTATION: The Pump Drivewill be programmed to include a countdown timer.

FEATURE: Rubber peg feet. IMPLEMENTATION: Rubber feet will be utilizedto ensure that the Pump Drive is stable and will not slide off a tableor shelf.

FEATURE: Custom housing with 30° platform. IMPLEMENTATION: A paintedaluminum custom enclosure will be utilized per an industrial design.

FEATURE: Pump drive to be private labeled. IMPLEMENTATION: Front andrear panel labeling will be specific color, text and contentrequirements.

FEATURE: Instruction Manual. IMPLEMENTATION: An instruction manual willbe supplied with the Pump Drive. In addition, this Pump Drive isdesigned to meet UL and cUL requirements to comply with UL

Mode of Operation—Continuous

Intended Use:

The systems described herein, such as the Autologous Platelet IntegratedConcentration (“APIC”) System is indicated for the rapid preparation ofautologous platelet rich plasma/platelet poor plasma from a small sampleof blood at the patient's point of care. The platelet richplasma/platelet poor plasma is mixed with autograft and/or allograftbone prior to application to a bony defect for improving bone grafthandling characteristics.

Product Specification

The following list is the design-input specifications for a Pump Driveof the system: Power Input: 100 VAC-240 VAC, 50/60 Hz; OperatingTemperature: 15° C.-35° C.; Shipping Test: ISTA 3A; Storage Temperature:−10° C.-65° C.; Humidity: 10%-90%; Speed Range: 232 rpm, (ref 3.1ml/sec.=186 ml/min.); Max. Load 30 psi (disposable); LineRegulation+/−2%; Load Regulation+/−2%; Speed Regulation+/−2%

Functional Description

The Pump Drive can provide flow (speed) at a rate of 3.1 ml/sec. (186ml/min). The flow rate can be programmed or factory set. The user canfollow the instructions for use to enable the Pump Drive. The Pump Drivecan be programmed to stop after 30 minutes of operation. A pump headinterlock circuit can remove power to the motor when the pump head rotorand rollers are exposed.

The Pump Drive can comprise the following main parts: a universal powersupply, 24 VDC permanent magnet DC, 5.9:1 gear motor w/encoder, a motordrive controller board, an Easy-Load II Pump Head, 2-Line LCD display,5-key Keypad and a two piece aluminum enclosure. The universal powersupply is a purchased part, which is designed and UL listed for medicalapplications. The power supply can be mounted on the rear of thechassis. The 24 VDC gear motor with encoder, are purchased as anassembled set. The motor/gearbox shaft will be coupled to the pump headshaft using a flexible coupler. The motor drive controller board islocated on the chassis. The EZ Load II Pump Head consists of a stainlesssteel rotor and rollers. On the back of the enclosure can be a medicalgrade, dual fused AC Inlet and the ON/OFF switch. The front of theenclosure comprises the Easy-Load II Pump Head, 2-Line LCD display & a5-key Keypad. The pump drive can be mounted on a table or shelf.

Product Operation

Place tube set into the pump head. Secure pump head cam latch.

Connect line cord to the AC entry module on the rear panel, connect linecord to the AC mains. Turn rear power switch to the ON position.ON/OFF—A switch mounted on the back panel of the enclosure. Switchespower from the mains to the universal power supply, which powers theelectronics and motor. KEYPAD—A 5-key keypad mounted on the front panel.START—Key press will enable the Pump Drive (motor operating). STOP—Keypress will disable the Pump Drive (motor stopped). ENTER—Key press willenable operator to confirm operator setting and sequence to the nextoperational display. YES—Key press will enable operator to confirmoperator setting and sequence to the next operational display. NO—Keypress will enable operator to confirm operator setting and sequence tothe next operational display. While the Pump is operational (running),all keys should be disabled except the STOP key.

Mechanical Inputs

Tubing selection—L/S size 16 tubing. ⅛″ ID×¼″ OD× 1/16″ wall. Ref: 0.8ml per rev.

Operator Related Outputs

LCD Display: A two-line LCD display will provide instructions and statusto the user. At power up the upper line will read: “APIC SYSTEM”“Version X.X” (X.X=the latest software revision). After the initialpower up display the display changes to: “PLACE TRAY” “PRESS ENTER.”After enter is pressed, display changes to: “INJECT PLASMA” “PRESSENTER.” After enter is pressed, display changes to: “LOAD TUBE” “LOCKPUMP LEVER.” Pump Head Lever is locked, (interlock is enabled); displaychanges to: “PRESS START.”

Pump starts to operate . . . display to indicate (time counting down)“30:00.” When time reaches 0:00, Pump Operation to stop, display toread: “CONCENTRATION DONE?” “PRESS YES OR NO.” If YES is pressed,display to read: “APIC READY.” If NO is pressed, display to read: “PRESSSTART” “TO CONTNUE.” After start is pressed, pump operation to start,display to indicate time counting down: “5:00.” When time reaches 0:00,Pump Operation to stop, display to read: “CONCENTRATION DONE?” “SELECTYES OR NO.” If YES is pressed, display to read: “APIC READY.” If NO ispressed—the 5 minute cycle repeats. The STOP key versus Pump Operations:The STOP key is always active. If STOP is pressed prior to initiatingpump cycle, display changes to: “CONTINUE SET UP?” “PRESS YES OR NO.” IfYES is pressed, display returns to display when STOP was pressed. If NOis pressed, display to read: “PLACE TRAY” “PRESS ENTER.” If STOP ispressed during pump cycle, display to read: “RESUME CYCLE?” “PRESS YESOR NO.” If YES is pressed, display to read: “PRESS START.” START ispressed, Pump operation resumes and display shows time of pressing“STOP” (XXX). NO is pressed, and display reads: “END CYCLE?” “PRESS YESOR NO.” YES is pressed, pump operation terminates and display reads: “000.” NO is pressed, pump operation resumes, and cycle repeats. The PumpHead—An L/S Easy Load II Pump Head comprises:

Single channel, fixed occlusion. 4-roller, stainless steel rotor.Compatible with L/S 16 size tubing, ref ⅛″ ID. Material: PolyphenyleneSulfide. Automated, spring loaded tubing retainers MECHANICAL OUTPUTS

Speed setting (rpm) 232; Max. Load continuous (psi) 30 (generated by thedisposable); Number of pump heads 1; Line Regulation: ±2; (% of MaxRPM); Load Regulation: ±2; (% of Max RPM); Speed Regulation ±2; (% ofMax RPM). “Load” is defined as the maximum load for which allspecifications apply, measured at the gearbox output. “Line Regulation”is defined as the change in speed when line voltage is changed fromnominal to minimum or nominal to maximum.

“Load Regulation” is defined as the change in speed when the load ischanged from nominal to zero or nominal to maximum.

Example 15—System Overview

The APIC PRP System (FIGS. 24-30) can contain three components forproducing APIC PRP; High Speed Bench Top Centrifuge; Peristaltic Pumpw/Custom Housing; and Disposables Kit for Collection, Separation, andAdministration of APIC PRP. The APIC System can separate and concentratea patient's own blood for therapeutic use by a physician. 60 cc to 120cc of a patient's blood can be drawn in to a collection bag, thentransferred to centrifuge tubes. The tubes can be centrifuged and therecovered plasma is then drawn off and transferred to a concentrationbag. The pump can circulate the blood through a Tangential Flow Filterconcentrating the APIC PRP down to a 5 cc to 10 cc of APIC. The APIC canthen be used by Physicians as they deem necessary and appropriate. Thesystem can include: Industry Standard Centrifuge and Peristaltic Pump,Private Labeled and Customized for APIC, Low Cost Disposable withFiltration, Majority of Disposable Components are PPS, Minimal number ofsteps. The system can include: Integrated Centrifuge and PumpSeparation, Custom Ergonomic Design, Lower Cost Equipment w/SmallerFootprint, Lower Cost and Less Disposables, Ease Of Use=Set It AndForget It

APIC Cell Free Concentration Kit: No Centrifugation; Direct Connectionof Blood Collection Bag to Concentration Bag; Two Filters

Example 16—In Vitro Cartilage Degradation Assay

To test the hypotheses that cartilage catabolism caused byproinflammatory cytokines and cartilage-degrading metalloproteinases(ADAMTS) can be inhibited by preparations of Leukocyte-rich PRP (LR-PRP)or Autologous Platelet Integrated Concentrate (APIC-PRP) a controlled invitro cartilage degradation assay was performed. BCE was treated withADAMTS-5, TNF-α or IL-1β in the presence or absence of LR-PRP orAPIC-PRP. Cartilage catabolism was measured following 2 or 3 days inculture by proteoglycan release via the presence of sulfatedglycosaminoglycan (sGAG) in the media. Bovine articular cartilageexplants (BCE, 200 tit mg) were isolated from 1-1.5 year-old heifers andare equilibrated 3 days in culture. BCE cultures were treated for 3 dayswith or without a 33% (v/v) Leukocyte rich platelet-rich Plasma(LR-PRP), blood, or APIC-PRP prepared from the same patient. Proteasedigestion of cartilage with 500 ng/ml ADAMTS-5 for 2 days was inhibitedwith a 2-fold serial dilution of APIC-PRP [ED₅₀=0.1% v/v]. Forcytokine-induced cartilage catabolism, BCE was incubated 3 days in SFMwith or without 80 ng/ml human TNF-α or 8 ng/ml human IL-1β. Cartilagedegradation was inhibited with the addition of 5 mg/ml A2M or 30% (v/v)APIC-PRP. To demonstrate a dose-response curve of APIC-PRP, 3-foldserial dilutions of APIC-PRP [ED50=3% v/v] were used to inhibitTNF-α/1L-1p induced cartilage degradation. Cartilage catabolism wasmeasured in culture supernatant by proteoglycan release via the presenceof sulfated glycosaminoglycan (sGAG) using a DMMB assay with chondroitinsulphate standard curve. Cartilage degradation in 200 mg BCE was inducedby addition of LR-PRP (33% v/v), demonstrating it as a source ofcartilage catabolism. Treatment with proinflammatory cytokines (80 ngimlTNF-α or 8 ng/ml IL-1β), ADAMTS-5 (500 ng/ml) also resulted in increasedsGAG in the medium. Addition of APIC-PRP inhibited cartilage catabolisminduced by cytokines, metalloproteinases or LR-PRP in a dose dependentmanner. The addition of LR-PRP at the highest concentration used in theAPIC-PRP study reduced but did not inhibit cartilage catabolism inducedby cytokines or MMP's measured by the release of sGAG in the medium(data not shown). Osteoarthritis (OA) is characterized by progressivedegeneration of articular cartilage. The BCE model is representative ofstudying putative therapeutics in OA. This study demonstrates thatLeukocyte-rich PRP (LR-PRP) contributed to cartilage catabolism, butAPIC-PRP protected cartilage from degradation by known OA mediators.This activity can be explained by the 5-10 fold increased concentrationof A2M in APIC-PRP over its concentration in blood. This conclusion isin agreement with experiments that demonstrate the protective effect ofA2M on cartilage. This improved understanding of cartilage biology andmetabolism should lead to clinical trials of APIC-PRP in humans.

Example 17—Chondroprotective Effect in Rabbit Model

The pathology ad osteoarthritis involves the upregulation ofinflammatory mediators and preleases such as matrix metalloproteases(MMPs) A2M is a naturally occurring plasma glycoprotein that is a potentprotease inhibitor. A2M is behaved to modulate cartilage catabolism byits ability to bind, trap and clear MMPs. Though A2M functionsthroughout multiple tissues and extracellular spaces, it does notnormally reach high levels within the intrarticular joint space. Theability of the Autologous Protease Inhibitor Concentrate (APIC-CellFree), which concentrates A2M from the blood, was tested to inhibitcartilage catabolism, and thereby attenuate the development ofosteoarthritis in a ACL-T rabbit model. The rabbit model represents afunctional load-bearing in vivo anatomical model for the evaluation ofosteoarthritis, which exhibits mechanical properties, morphologicalstructures, and healing capacity similar to human tissues. Female 8-12months old New Zealand white rabbits were used in this study. Thisrabbit model represents a functional load-bearing in vivo anatomicalmodel for the evaluation of osteoarthritis which exhibits mechanicalproperties, morphological structures and healing capacity similar tohuman tissue. Multiple Injection Cohort (Group 1): 6 rabbits receivedACL-T surgery on the right knee and sham surgery on the left knee. Fourinjections of 0.3 mL Autologous Protease Inhibitor Concentrate(APIC-Cell Free) were prepared from the rabbit blood and wereadministered on day 1. 4, 14, and 28 following the ACL knee injury.Rabbits received an equivalent volume of the sterile isotonic saline inthe contra-lateral control knee. The rabbits were monitored for 6 weeks,then sacrificed for cartilage degeneration assessment. Control Group(Group 2): 6 rabbits received ACL-T surgery on the right knee withoutsham surgery on the left knee. These rabbits were the control group andaccordingly did not receive any treatment.

Autologous A2M Concentrate Preparation

Prior to the ACL injury, 20 mL of blood was removed from each animal ingroup 1 and used to prepare the APIC Cell Free concentrate using aseries of filters. Every rabbit received the protease inhibitorconcentrate from its own blood. Six weeks after the ACL-T operation theanimal was sacrificed for macroscopic and microscopic knee jointcartilage evaluation to determine OA progression

Macroscopic and Histological Analyses

For macroscopic evaluation, the distal femoral condyles and tibialplateau surfaces were analyzed and lesions were classified using avalidated 0 to 8 scale as previously described. The locations of thelesions in the joint were recorded by a specific nine-area grid of eachjoint surface, following the classification of the InternationalCartilage Repair Society (OARSI), which was adapted to the rabbit kneeby Lindhorst et al. After macroscopic examination. Isolated femoral andtibial samples were feed and decalcified for histological (microscopicevaluation). Macroscopic evaluation of the femur and tibia demonstratedfeatures consistent with cartage catabolism consistent with OA.Treatment with APIC Cell Free considerably improved cartilageappearance, similar to the sham surgery control (FIGS. 12-14).Application of APIC reduced cartilage degradation by 53+/−20% comparedto untreated controls (mean±SEM. p=0.0086) (FIGS. 13A and B). Theconcentration of A2M in the APIC Cell Free varied front 5-65 mg/ml.There was a dose-dependent correlation between higher concentrations ofA2M in the API Cell Free and decreased OARSI total knee score on themacroscopic evaluation (FIGS. 13A and B). There was also adose-dependent therapeutic benefit to APIC Cell Free treatment observedin sum OARSI histopathology evaluations of Safarin-O staining (r²=0.73),Structure (r²=0.76), Chondrocyte density (r²=0.50), and ClusterFormation (r²=0.97) (FIG. 14). The data suggests that the AutologousProtease inhibitor Concentrate (APIC-Cell Free), which contains 9-10times the A2M concentration in blood, has a chondroprotective effect onan osteoarthritis rabbit model.

Example 18—Effect of A2M on BCEs

To test the hypothesis that the addition of proinflammatory cytokines orcartilage-degrading metalloproteinases (ADAMTS and MMP) stimulatecartilage degradation that will be inhibited by A2M, a controlled invitro cartilage degradation assay was performed. Bovine CartilageExplants (BCE) were treated with or without proinflammatory cytokines(TNF-α or IL-1β) or cartilage-degrading metalloproteinases (ADAMTS-5,ADAMTS-4, MMP-7, or MMP-12) in the presence or absence of purified A2M.

Bovine articular cartilage explants (BCE. 100±4 mg) were isolated from1-1.5 year-old heifers and were equilibrated 3 days in culture. Todegrade cartilage by protease digestions, BCE was incubated 2 days inSerum-free Media (SFM) with or without 500 ng/mL ADAMTS-4 or ADAMTS-5and 3-5 μg/mL of MMP-3, MMP-7, MMP-12, or MMP-13. MMP-3 was activatedwith chymotrypsin before application on BCE. For cytokine-inducedcartilage catabolism, BCE (200+/−4 mg) was incubated 3 days in SFM withor without 80 ng/ml human TNF-α and 8 ng/mL human1L-1β. Cartilagedegradation was inhibited with the addition of 100 μg/mL of purifiedhuman A2M for protease digestion or 5 mg/mL A2M for cytokine-induceddegradation.

Cartilage catabolism was measured in culture supernatant by 1)proteoglycan release via the presence of sulfated glycosaminoglycan(sGAG) and 2) the presence of cartilage proteoglycan fragments byBio-Rad Stainless SDS-PAGE and Aggrecan G3 fragments by Westernblotting.

Fibronectin and Aggrecan Complexes (FAC) were formed by combiningdegraded cartilage matrix proteoglycans from the BCE experiments withFibronectin and Synovial Fluid and incubating for 4 hours. Newly formedFAC was measured by the FACT ELI SA, with the alteration of using anα-Aggrecan G3 antibody needed to recognize bovine aggrecan.

The IC₅₀ needed to inhibit cartilage catabolism by 500 mg/mL proteaseswas 7 μg/mL A2M for ADAMTS-5 and 3 μg/mL for ADAMTS-4. Addition of 5mg/mL A2M also inhibited cartilage catabolism induced by TNF-α or IL-1β.Further, A2M blocked production of Aggrecan G3 fragments, which formcomplexes with fibronectin and are a marker for pain and degradingjoints. (FIGS. 7-10).

1.-202. (canceled)
 203. A composition comprising a recombinant A2Mpolypeptide comprising a non-natural bait region, wherein thenon-natural bait region comprises a sequence with at least 6 contiguousamino acid residues of a sequence selected from the group consisting ofSEQ ID NOs: 5-9, 11, 17, 27-29, 34-35, 37, 39-42, 45-47 and 50-66. 204.The composition of claim 203, wherein the non-natural bait regioncomprises a sequence with at least 80% identity to at least 18contiguous amino acid residues of a sequence selected from the groupconsisting of SEQ ID NOs: 47, 52-53, 55, 61-63 and
 66. 205. Thecomposition of claim 203, wherein the non-natural bait region comprisesa sequence with at least 90% identity to at least 25 contiguous aminoacid residues of SEQ ID NO:
 47. 206. The composition of claim 203,wherein the non-natural bait region comprises a sequence with at least24 contiguous amino acid residues of SEQ ID NO:
 47. 207. The compositionof claim 203, wherein the non-natural bait region comprises two or moreprotease recognition sequences.
 208. The composition of claim 203,wherein the recombinant A2M polypeptide is characterized by an enhancedinhibition of a protease compared to an inhibition of the protease by awild-type A2M protein, wherein the protease is selected from the groupconsisting of a serine protease, a threonine protease, a cysteineprotease, an aspartate protease, a metalloprotease, a glutamic acidprotease, and combinations thereof.
 209. The composition of claim 208,wherein the enhanced inhibition is nonspecific to the protease.
 210. Thecomposition of claim 208, wherein the enhanced inhibition is specific tothe protease.
 211. The composition of claim 203, wherein the recombinantA2M polypeptide further comprises an abnormal glycosylation site. 212.The composition of claim 203, wherein the recombinant A2M polypeptidehas a longer half-life than a half-life of a wild type A2M protein whendisposed within a subject.
 213. The composition of claim 203, whereinthe non-natural bait region comprises a consensus sequence for aprotease selected from the group consisting of a serine protease, athreonine protease, a cysteine protease, an aspartate protease, ametalloprotease, a glutamic acid protease, and combinations thereof.214. The composition of claim 203, wherein the non-natural bait regioncomprises a consensus sequence for a protease selected from the groupconsisting of a matrix metalloproteinase (MMP); a disintegrin andmetalloproteinase with thrombospondin motifs (ADAMTS), chymotrypsin,trypsin, elastase, a compliment factor, a clotting factor, thrombin,plasmin, subtilisin, neprilysin, procollagen peptidase, thermolysin,pregnancy-associated plasma protein A, bone morphogenetic protein 1,lysostaphin, insulin degrading enzyme, ZMPSTE2, ZMPSTE4, ZMPSTE24,acetylcholinesterase, and combinations thereof.
 215. The composition ofclaim 203, wherein the non-natural bait region comprises a proteaserecognition sequence from a non-A2M protein.
 216. The composition ofclaim 203, wherein the one or more protease recognition sequencescomprise a protease recognition sequence comprising a suicide inhibitor,wherein the suicide inhibitor is operable to covalently attach aprotease to the recombinant A2M polypeptide.
 217. The composition ofclaim 203, wherein the recombinant A2M polypeptide comprises a sequencewith at least 80% identity to SEQ ID NO:
 4. 218. The composition ofclaim 203, wherein the wild-type A2M polypeptide comprises the sequenceof SEQ ID NO:
 3. 219. A composition comprising an isolated recombinantA2M polynucleotide, wherein the recombinant A2M polynucleotide encodesfor a non-natural bait region, wherein the non-natural bait region has asequence with at least 6 contiguous amino acid residues of a sequenceselected from the group consisting of SEQ ID NOs: 5-9, 11, 17, 27-29,34-35, 37, 39-42, 45-47 and 50-66.
 220. The composition of claim 219,wherein the non-natural bait region comprises one or more proteaserecognition sequences.
 221. The composition of claim 219, wherein thenon-natural bait region has a sequence with at least 80% identity to atleast 18 contiguous amino acid residues of a sequence selected from thegroup consisting of SEQ ID NOs: 47, 52-53, 55, 61-63 and
 66. 222. Thecomposition of claim 219, wherein the non-natural bait region has asequence with at least 90% identity to at least 25 contiguous amino acidresidues of SEQ ID NO: 47.